High-purity steviol glycosides

ABSTRACT

Methods of preparing highly purified steviol glycosides, particularly Rebaudioside D, are described. The methods include purification from the extraction stage of the Stevia rebaudiana Bertoni plant, purification of steviol glycoside mixtures, Rebaudioside D and Rebaudioside A from a commercial Stevia extract, and purification of Rebaudioside D from remaining solutions obtained after isolation and purification of Rebaudioside A and a high purity mixture of steviol glycosides. The methods are useful for producing high purity Rebaudioside D, Rebaudioside A, and steviol glycoside mixtures. The high purity steviol glycosides are useful as non-caloric sweeteners in edible and chewable compositions such as any beverages, confectioneries, bakery products, cookies, and chewing gums.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/313,375, filed on Mar. 12, 2010, U.S. ProvisionalPatent Application No. 61/313,388, filed on Mar. 12, 2010, U.S.Provisional Patent Application No. 61/373,491, filed on Aug. 13, 2010,and U.S. Provisional Patent Application No. 61/385,215, filed on Sep.22, 2010, the contents of which applications are incorporated herein byreference in their entireties.

TECHNICAL FIELD

The present invention relates to a process for the treatment of varioussteviol glycoside sources in order to isolate purified sweet glycosides,either as a mixture or as individual steviol glycosides such asRebaudioside A or Rebaudioside D.

BACKGROUND OF THE INVENTION

High intensity sweeteners possess a sweetness level that is many timesgreater than the sweetness level of sucrose. They are essentiallynon-caloric and are used widely in the manufacturing of diet and reducedcalorie food. Although natural caloric sweetener compositions, such assucrose, fructose, and glucose, provide a desirable taste to consumers,they have a caloric content. High intensity sweeteners, beingessentially non-caloric, do not affect the blood glucose level andprovide little or no nutritive value.

However, the high intensity sweeteners generally used as sugar (sucrose)substitutes possess taste characteristics different from those of sugar.The taste characteristics that differ from those of sugar may includethe temporal profile of the sweet taste, maximal response, flavorprofile, mouthfeel, and adaptation behavior. For example, the sweettaste of some high-potency sweeteners are slower in onset and longer induration than the sweet taste produced by sugar and thus change thetaste balance of a food composition. Because of these differences, theuse of high-potency sweeteners to replace a bulk sweetener such assugar, in a food or beverage, may cause an imbalance in the temporaland/or flavor profile. If the taste profile of high-potency sweetenerscould be modified to impart desired taste characteristics, high-potencysweeteners could be used to provide more desirable taste characteristicsto low calorie beverages and food products.

On the other hand, high-potency sweeteners may have some cost andfunctional advantages compared to sugar. There is significantcompetition among sugar and non-sugar sweeteners in the soft drinkindustry, in countries where the use and production of high-potencysweeteners is permitted, and in countries with overvalued sugar prices.

At present high intensity sweeteners are used worldwide. They can beboth synthetic and natural origin.

Examples of synthetic sweeteners include, but are not limited to,sucralose, potassium acesulfame, aspartame, alitame, saccharin,neohesperidin dihydrochalcone, cyclamate, neotame, dulcin, suosan,N—[N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester,N—[N-[3-(3-hydroxy-4-methoxyphenyl)-3-methylbutyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester,N—[N-[3-(3-methoxy-4-hydroxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester, salts thereof, and the like.

Examples of natural high intensity sweeteners include, but are notlimited to, Stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C,Rebaudioside E, Rebaudioside F, Steviolbioside, Dulcoside A, Rubusoside,mogrosides, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acidand its salts, thaumatin, perillartine, pernandulcin, mukuroziosides,baiyunoside, phlomisoside−1, dimethyl-hexahydrofluorene-dicarboxylicacid, abrusosides, periandrin, carnosiflosides, cyclocarioside,pterocaryosides, polypodoside A, brazilin, hernandulcin, phillodulcin,glycyphyllin, phlorizin, trilobatin, dihydroflavonol,dihydroquercetin-3-acetate, neoastilibin, trans-cinnamaldehyde, monatinand its salts, selligueain A, hematoxylin, monellin, osladin,pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin,curculin, neoculin, chlorogenic acid, cynarin, Luo Han Guo sweetener,and siamenoside.

High intensity sweeteners can be derived from the modification ofnatural high intensity sweeteners by, for example, fermentation, contactwith enzymes, or derivatization.

At present about twelve high intensity sweeteners are used worldwide.These are acesulfame-K, alitame, aspartame, cyclamate, glycyrrhizin,neohesperidin dihydrochalcone (NHDC), saccharin, Stevioside, sucralose,thaumatin, neotame, and Rebaudioside A.

The high intensity sweeteners can be grouped into a few differentgenerations. The first generation, represented by cyclamate,glycyrrhizin and saccharin, has a long history of use in food. Thesecond generation includes acesulfame-K, aspartame, NHDC and thaumatin.Alitame, neotame, sucralose, Stevioside, and Rebaudioside A belong tothe third generation.

The standard sweetening power associated with each high intensitysweetener is given in TABLE 1. However, when they are used in blends,the sweetening power can change significantly.

TABLE 1 Sweetener Sweetness power Saccharose (sucrose) 1 Acesulfame-K200 Alitame 2000 Aspartame 200 Cyclamate 30 Glycyrrhizin 50 NHDC 1000Saccharin 300 Stevioside 200 Rebaudioside A 450 Thaumatin 3000 Sucralose600

“Natural” and “organic” foods and beverages have become the “hottestarea” in the food industry. The combination of consumers' desire,advances in food technology, and new studies linking diet to disease anddisease prevention have created an unprecedented opportunity to addresspublic health through diet and lifestyle.

A growing number of consumers perceive the ability to control theirhealth by enhancing their current health and/or hedging against futurediseases. This creates a demand for food products with enhancedcharacteristics and associated health benefits, and creates a food andconsumer market trend towards a “whole health solutions” lifestyle. Theterm “natural” is highly emotive in the world of sweeteners and has beenidentified as a term that is highly trusted by consumers, along with“whole grain”, “heart-healthy” and “low-sodium”. Among many consumers,the term “natural” is closely related to “healthier”. In this respect,natural high intensity sweeteners can have better commercial potentialthan artificial sweeteners.

Stevia rebaudiana Bertoni is a perennial shrub of the Asieraceae(Composilae) family native to certain regions of South America. Theleaves of the plant contain diterpene glycosides in an amount rangingfrom about 10 to 20%. These diterpene glycosides are about 150 to 450times sweeter than sugar. The leaves of the Stevia rebaudiana Bertoniplant have been traditionally used for hundreds of years in Paraguay andBrazil to sweeten local teas and medicines. The plant is commerciallycultivated in Japan, Singapore, Taiwan, Malaysia, South Korea, China,Israel, India, Brazil, Australia, and Paraguay.

At present more than 230 Stevia species have been discovered, includingone Stevia species that has significant sweetening properties. The planthas been successfully grown under a wide range of conditions from itsnative subtropics to the cold northern latitudes.

The extract of the Stevia rebaudiana plant contains a mixture ofdifferent sweet diterpene glycosides which have a single base, steviol,and differ by the presence of carbohydrate residues at positions C13 andC19. These glycosides accumulate in Stevia leaves and composeapproximately 10%-20% of the total dry weight. Typically, on a dryweight basis, the four major glycosides found in the leaves of Steviaare Dulcoside A (0.3%), Rebaudioside C (0.6-1.0%), Rebaudioside A (3.8%)and Stevioside (9.1%). Other glycosides identified in Stevia extractinclude Rebaudioside B, C, D, E, and F, Steviolbioside and Rubusoside.Among steviol glycosides only Stevioside and Rebaudioside A areavailable on a commercial scale.

Steviol glycosides have zero calories and can be used wherever sugar isused. They are ideal for diabetic and low calorie diets. In addition,the sweet steviol glycosides possess functional and sensory propertiessuperior to those of many high potency sweeteners.

The chemical structures of the diterpene glycosides of Stevia rebaudianaBertoni are presented in FIGS. 2a -2 k.

The physical and sensory properties of steviol glycosides are wellstudied only for Stevioside and Rebaudioside A. Stevioside is about 210times sweeter than sucrose, and Rebaudioside A is between 200 and 400times sweeter than sucrose. Rebaudioside A is considered to have themost favorable sensory attributes of the four major steviol glycosides.

Amongst the sweet diterpenoid glycosides of Stevia, Rebaudioside D hasbeen identified as the least bitter, and with the least persistentaftertaste. Impurities can significantly increase the bitterness ofditerpenoid glycosides.

The glycosides can be extracted from leaves using either water ororganic solvent extraction. Supercritical fluid extraction and steamdistillation methods have also been described. Methods for the recoveryof diterpene sweet glycosides from Stevia rebaudiana using supercriticalCO₂, membrane technology, and water or organic solvents, such asmethanol and ethanol, may also be used.

Generally the production of an extract includes extraction of plantmaterial with water or a water-organic solvent mixture, precipitation ofhigh molecular weight substances, deionization, and decolorization,purification on specific macroporous polymeric adsorbents, concentrationand drying.

All of the existing methods for the recovery of steviol glycosidesinvolve the isolation and purification of a steviol glycoside from theinitial plant extract, and do not include a method for the furthertreatment of residual solutions or the purification of minor compounds.Thus, there is a need for an efficient and economical method for thecomprehensive retreatment of extract produced from the Stevia rebaudianaBertoni plant.

Due to regulatory requirements, only materials containing more than 95%total steviol glycosides are allowed to be used as sweeteners for humanconsumption. The majority of commercially available Stevia rebaudianaraw extracts contain 80-90% total steviol glycosides. Hence furtherpurification is necessary to obtain highly purified products with morethan 95% total steviol glycoside content. Therefore, there is a need fora commercially viable process for enhancing steviol glycoside content inlow purity steviol glycoside preparations, to the levels which allowtheir usage in food.

Among sweet glycosides existing in Stevia rebaudiana, only Steviosideand Rebaudioside A are available at moderate cost at <80% purity and athigh cost at >80% purity. There are no commercial quantities ofRebaudiosides B, D, E, F and C available in the market.

Rebaudioside D (CAS No: 63279-13-0) is one of the sweet glycosides foundin Stevia rebaudiana. Its isolation and purification is a verychallenging task due to its low content in Stevia leaves. The averageRebaudioside D content in dry leaves ranges from about 0.01-0.20%.Moreover, many analytical techniques often fail to detect Rebaudioside Din Stevia leaves or steviol glycoside preparations, due to its lowcontent.

Only recently have highly purified Rebaudioside D reference materialsbecome available on the market. Due to the high cost of the purificationtechniques employed, the price of such materials makes it impractical touse them in any area other than analytical chemistry.

However, studies show that highly purified forms of Rebaudioside Dpossess a very desirable taste profile, almost lacking in bitterness andin the lingering licorice aftertaste typical for other steviolglycosides. These properties multiply the significance of Rebaudioside Dand attract great interest for methods of preparation of highly purifiedforms of Rebaudioside D.

The methods of Rebaudioside D preparation described in the literatureemploy costly chromatographic techniques which are only applicable forlaboratory or pilot scale production. There is no published data on thecommercial isolation and purification of Rebaudioside D.

Sakamoto et al. describe a process of isolation of rebaudioside D fromthe glycosidic fraction of stevia leave methanolic extract preparedaccording to Kohda et al. Sakamoto I., Yamasaki Tanaka O. (1977),“Application of ¹³C NMR Spectroscopy to Chemistry of NaturalGlycosides:Rebaudioside-C, a New Sweet Diterpene Glycoside of Steviarebaudiana.” Chem. Pharm. Bull., 25(4), p. 844; Kohda H., Kasai R.,Yamasaki K., Murakami K., Tanaka O. (1976), “New sweet diterpeneglucosides from Stevia rebaudiana.” Phytochemisty, 15, p. 981. Theprocess comprises recrystallization of a glycosidic fraction frommethanol and further chromatography on silica gel. The described processemploys solvent extraction and chromatographic techniques which areuseful in laboratory and pilot scale, but have limited scale-uppotential due to the high cost of the process and toxicity of theextraction solvents.

Hence, there is a need for a simple, efficient, and economical methodfor the production of high purity Rebaudioside D.

There is also a need for a commercially viable process for enhancingsteviol glycoside content in low purity steviol glycoside preparationsto the levels which allow their usage in food.

Any technological scheme which can effect the purification of a mixtureof low purity steviol glycosides, into a mixture of highly purifiedsteviol glycosides and highly purified Rebaudioside D, will have acertain advantage over the techniques currently known in the art.

SUMMARY OF THE INVENTION

The present invention provides a method for the preparation of highlypurified mixtures of steviol glycosides, or individual glycosides, fromlow purity steviol glycoside mixtures, from Stevia extracts, and fromthe Stevia rebaudiana Bertoni plant, and the products resulting from themethod.

An object of the invention is to provide an efficient method forisolating and purifying different steviol glycosides, particularlyRebaudioside D, from various mixtures of steviol glycosides, from Steviaextracts, and from the Stevia rebaudiana Bertoni plant, and a highpurity Rebaudioside D resulting from this method.

The present invention is directed to a method for purifying steviolglycosides, which includes passing a solution of steviol glycosidesthrough a multi-column system including a plurality of columns packedwith an adsorbent resin, to obtain at least one column having adsorbedsteviol glycosides. The method further includes removing impurities fromthe multi-column system. The adsorbed steviol glycosides are then elutedfrom the column or columns having adsorbed steviol glycosides, to obtainan eluted solution of steviol glycosides. The eluted solution of steviolglycosides may then be dried to obtain a purified mixture of steviolglycosides.

The present invention is also directed to a method for purifyingRebaudioside D, which includes passing a solution of steviol glycosidesthrough a multi-column system including a plurality of columns packedwith an adsorbent resin, to obtain at least one column having adsorbedsteviol glycosides. The method further includes removing impurities fromthe multi-column system. Fractions with a high Rebaudioside D contentare then eluted with an aqueous alcohol solution. Rebaudioside D may befurther purified by mixing the high Rebaudioside D fractions with afirst aqueous alcohol solution to obtain a Rebaudioside D solution,inducing crystallization to obtain first crystals of Rebaudioside D, andseparating the first crystals of Rebaudioside D from the Rebaudioside Dsolution, wherein the first crystals have a purity level greater thanabout 60% (w/w) on a dry basis. The first crystals may then be suspendedin a second aqueous alcohol solution, to obtain second crystals ofRebaudioside D and a third aqueous alcohol solution. The second crystalsof Rebaudioside D may be separated from the third aqueous alcoholsolution. These second crystals may have a purity level greater thanabout 95% (w/w) on a dry basis.

In accordance with the present invention, the solution of steviolglycosides which is passed through a multi-column system may be preparedfrom a biomass of the Stevia rebaudiana Bertoni plant. To obtain asolution of steviol glycosides from a biomass of the Stevia rebaudianaBertoni plant, a crude extract may be produced by contacting the biomasswith water, separating insoluble material from the crude extract toobtain a filtrate containing steviol glycosides, and treating thefiltrate to remove high molecular weight compounds and insolubleparticles to obtain the solution of steviol glycosides.

The solution of steviol glycosides prepared from a biomass of the Steviarebaudiana Bertoni plant may be passed through a multi-column systemincluding a plurality of columns packed with an adsorbent resin, toobtain at least one column having adsorbed steviol glycosides.Impurities may be removed from the multi-column system by eluting themulti-column system with a washing solution. The adsorbed steviolglycosides are then eluted from the column or columns having adsorbedsteviol glycosides, to obtain an eluted solution of steviol glycosides.The eluted solution of steviol glycosides may be further purified, usinga method including treating the eluted solution to obtain a decolorizedsolution, evaporating the solvent from the decolorized solution toobtain a first adsorption solution, passing the first adsorptionsolution through a column with a macroporous adsorbent to obtain asecond adsorption solution, deionizing and concentrating the secondadsorption solution, and drying the deionized and concentrated secondadsorption solution to obtain a purified steviol glycoside mixture withat least about 95% by weight total steviol glycosides.

Purified steviol glycoside mixtures and purified individual steviolglycosides, prepared in accordance with the present invention, may beused in a variety of products including, but not limited to, foods,beverages, pharmaceutical compositions, tobacco products, nutraceuticalcompositions, oral hygiene compositions, and cosmetic compositions.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of the invention. The drawings illustrate embodiments ofthe invention and together with the description serve to explain theprinciples of the embodiments of the invention.

FIG. 1 shows the structure of steviol glycosides in the Steviarebaudiana Bertoni leaves.

FIGS. 2a-2k show the chemical structures of Stevia rebaudiana Bertoniglycosides.

FIG. 3 shows the structure of Rebaudioside D.

FIG. 4 is a diagram illustrating the purification of Rebaudioside D fromthe extraction stage.

FIG. 5 is a diagram illustrating the isolation of Rebaudioside Dfractions from the portion of a Stevia extract adsorbed on a macroporousresin.

FIG. 6 is a diagram illustrating the isolation of Rebaudioside Dfractions from the portion of a Stevia extract which was not adsorbed ona macroporous resin (i.e. the water fraction).

FIG. 7 is a diagram illustrating a one-stage purification scheme ofRebaudioside A using ethanol-water systems, followed by the isolation ofRebaudioside D from a remaining solution.

FIG. 8 includes HPLC charts of Rebaudioside D at various stages ofpurification.

FIG. 9 is a diagram illustrating a purification scheme of Rebaudioside Dalong with Rebaudioside A, using ethanol at the first stage.

FIG. 10 is a diagram illustrating the isolation of Rebaudioside D from aremaining solution after the one-stage purification of Rebaudioside A,using methanol as solvent.

FIG. 11 is a diagram illustrating the separation of high and lowRebaudioside D fractions from part of a remaining solution afterisolation of Rebaudioside A adsorbed on macroporous resin.

FIG. 12 is a diagram illustrating the separation of high and lowRebaudioside D fractions from part of a remaining solution afterisolation of Rebaudioside A which was not adsorbed on macroporous resin(i.e. the water fraction).

FIG. 13 is a diagram illustrating the isolation of Rebaudioside D from aremaining solution after a three-stage purification of Rebaudioside A.

FIG. 14 is a diagram illustrating the isolation of Rebaudioside D from aremaining solution after a two-stage purification of Rebaudioside A bycrystallization.

FIG. 15 is a diagram illustrating the purification of Rebaudioside Dfrom a remaining solution after a two-stage purification of RebaudiosideA by washing.

FIG. 16 is an FTIR spectrum of Rebaudioside D.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for the production of highlypurified mixtures of steviol glycosides and of highly purifiedindividual steviol glycosides, such as Rebaudioside D, by variousschemes.

The production scheme and priority of the steps can be changed dependingon the main goal of the process and the desired business andtechnological model.

Highly purified steviol glycoside mixtures or individual steviolglycosides, alone or in combination with other sweeteners and/or otheringredients, are useful as non-caloric sweeteners in edible and chewablecompositions, such as any beverages, confectioneries, bakery products,cookies, chewing gums, oral hygiene compositions, and the like.

Hereinafter the term “steviol glycoside(s)” will mean Rebaudioside A,Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E,Rebaudioside F, Stevioside, Steviolbioside, Dulcoside A, Rubusoside, orother glycosides of steviol and combinations thereof.

Hereinafter the term “TSG content” will mean Total Steviol Glycosidescontent, and it will be calculated as the sum of the content of allsteviol glycosides on a dry basis, including Rebaudioside A,Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E,Rebaudioside F, Stevioside, Steviolbioside, Dulcoside A and Rubusoside.

Hereinafter the term “highly purified” or “high purity” will mean asteviol glycoside purity and/or TSG level of at least 95% (w/w) on a drybasis.

Hereinafter the term “low purity” will mean a steviol glycoside purityand/or TSG level of less than 95% (w/w) on a dry basis.

Hereinafter the terms “Reb A”, “Reb B”, “Reb C”, “Reb E”, and “Reb F”refer to Rebaudiosides A, B, C, E, and F.

Hereinafter the term “Reb D” refers to Rebaudioside D (CAS No.63279-13-0).

Hereinafter the term “impurity” will mean any compound other thansteviol glycosides which are present in the mixture at more than 0.0001%(w/w) on a dry basis. Examples of impurities include, but are notlimited to, typical plant materials, such as pigments and saccharides,phenolic compounds, volatile oil components, sterols, triterpenes,flavonoids, coumarins, non-glycosidic diterpenes (sterebins)spathulenol, decanoic acid, 8,11,14-ecosatrienoic acid,2-methyloctadecane, pentacosane, octacosane, stigmasterol, bsitosterol,a- and b-amyrine, lupeol, b-amyrin acetate, and pentacyclic triterpene.

In one embodiment of this invention a column system with the followingparameters was used. A system of consecutive (serial) or parallelconnected columns, of the same or different volumes, packed with asorbent with different affinities to impurities and steviol glycosides.When the system is used in parallel connection mode, the inlet of eachcolumn may connect to a separate feed source while the outlet of eachcolumn connects to a separate receiver. The following description willhighlight the mode of column connection specific to each particularcase. Within the same stage the system may function as an entity ofseveral parallel and serial connected column groups and separatecolumns. The number of columns may be 3-15. The ratio of the volume ofthe first column to the volume of the second column is preferably in therange of about 1:1 to 1:10. The ratio of the volume of the last columnto the volume of the previous, or penultimate, column is preferably inthe range of about 3:1 to 1:10. The columns may be packed with sorbentup to about 75-100% of their total volume. The columns may be maintainedat a temperature in the range of about 5-80° C., and preferably in therange of about 15-25° C.

The alcohols employed in the invention may be selected from the groupconsisting of alkanols, and are preferably selected from the groupincluding methanol, ethanol, n-propanol, 2-propanol, 1-butanol, and2-butanol.

The sorbents may be any macroporous polymeric adsorption resins capableof adsorbing steviol glycosides, such as the Amberlite® XAD series (Rohmand Haas), Diaion® HP series (Mitsubishi Chemical Corp), Sepabeads® SPseries (Mitsubishi Chemical Corp), Cangzhou Yuanwei YWD series (CangzhouYuanwei Chemical Co. Ltd., China), or the equivalent.

In one embodiment of the present invention, an aqueous or aqueousalcoholic solution of a low purity steviol glycoside mixture was passedthrough the consecutively connected column system. If an aqueousalcoholic solution is used, the water to alcohol ratio (vol/vol) in theaqueous alcoholic solution may be in the range of about 99.9:0.1 to60:40. As a result impurities and different steviol glycosides areretained in different sections of the column system. Impurities withhigher affinities to the sorbent are retained in the first column,impurities with lower affinities to the sorbent are retained in the lastcolumn, and different steviol glycosides are retained in differentsections of the system at different concentrations, depending on theiraffinities to the sorbent. As a result, an initial mixture of low puritysteviol glycosides separates into different portions retained ondifferent columns. The portions differ from each other both by TSG andindividual glycoside (particularly Reb D) content.

In another embodiment of the present invention, columns with retainedsteviol glycosides are subjected to elution of residual impurities inorder to increase further the purity level of the final products.Columns are subsequently eluted with an aqueous or aqueous alcoholicsolution of an acid, an aqueous or aqueous alcoholic solution of a base,and an aqueous alcoholic solution, in a specific order. The water toalcohol ratio (vol/vol) in the aqueous alcoholic solution is in therange of about 99.9:0.1 to 60:40. The elution of impurities is carriedout either from each column separately (parallel connection) or from twoor more consecutively (serially) connected columns.

Another embodiment of the present invention comprises the elution ofcolumns with an aqueous alcoholic solution in which the water to alcoholratio (vol/vol) is in the range of about 60:40 to 0.1:99.9. The elutionis carried out either from each column separately (parallel connection)or from two or more consecutively (serially) connected columns.

One embodiment of the present invention utilizes centrifugationtechniques to separate crystals or other suspended solids from a liquid.However any other type of equipment which separates a precipitate from aliquid, such as various types of centrifuges or filtration systems, canbe used as well.

Another embodiment utilizes a rotary vacuum dryer to dry separatedcrystals and/or solids. However different type of dryers such as fluidbed dryers, rotary tunnel dryers, or plate dryers are suitable as well.

Exemplary embodiments of this invention are described in detail belowand illustrated in FIGS. 4-16.

Purification of Steviol Glycosides from a Low Purity Steviol GlycosidesMixture

The present invention is directed to a method for preparing a purifiedsteviol glycoside mixture from a steviol glycoside mixture of lowerpurity, including the steps of providing a steviol glycoside mixture,dissolving the steviol glycosides mixture in a solvent to produce asolution of steviol glycosides, passing the solution of steviolglycosides through a multi-column system including a plurality ofcolumns packed with an adsorbent resin, eluting impurities from themulti-column system with water or an aqueous solution, eluting steviolglycoside mixture fractions from the multi-column system with an aqueousalcohol solution, and drying the steviol glycoside mixture fractions toobtain the purified steviol glycoside mixture.

According to the present invention the purification of steviolglycosides was developed from a steviol glycosides mixture with a TSGcontent of less than 95% w/w on a dry basis.

In one example, the TSG content of a “low purity” initial mixture was85.29% (w/w on a dry basis), comprising 0.16% Rubusoside, 0.68%Dulcoside A, 26.20% Stevioside, 9.78% Rebaudioside C, 1.86% RebaudiosideF, 45.92% Rebaudioside A, 0.51% Rebaudioside D, 0.09% Steviolbioside,and 0.09% Rebaudioside B.

A column system as described above was used to purify the “low purity”initial mixture of steviol glycosides in a four-stage process, which isdiscussed below.

The first stage of purification of the steviol glycoside mixturecomprises passing an aqueous or aqueous alcoholic solution of a lowpurity steviol glycoside mixture through the consecutively connectedcolumn system. The water to alcohol ratio (vol/vol) in the aqueousalcoholic solution is in the range of 99.9:0.1 to 60:40. As a result,impurities and different steviol glycosides are retained in differentsections of the column system. Impurities with higher affinities to thesorbent are retained in the first column, impurities with loweraffinities to the sorbent are retained in the last column, and differentsteviol glycosides are retained in different sections of the system atdifferent concentrations, depending on their affinities to sorbent. Thesteviol glycosides are mostly retained in the middle section of thesystem.

In one example, the total elution of the middle section columns afterthe first stage resulted in a steviol glycoside mixture with a TSGcontent of 91.14% (w/w on a dry basis) comprising 0.17% Rubusoside,0.72% Dulcoside A, 28.05% Stevioside, 10.43% Rebaudioside C, 1.98%Rebaudioside F, 49.07% Rebaudioside A, 0.54% Rebaudioside D, 0.09%Steviolbioside, and 0.09% Rebaudioside B.

In the second stage of purification, the middle section columns withretained steviol glycosides are subjected to elution of residualimpurities which still remain after the first stage of separation, inorder to increase further the purity level of the final products.Columns are subsequently eluted with an aqueous or aqueous alcoholicsolution of an acid, an aqueous or aqueous alcoholic solution of a base,and an aqueous alcoholic solution, in a specific order. The water toalcohol ratio (vol/vol) in the aqueous alcoholic solution is in therange of about 99.9:0.1 to 60:40. Elution of impurities is carried outeither from each column separately (parallel connection) or from two ormore consecutively (serially) connected columns.

In one example, the total elution of middle section columns after thesecond stage resulted in a steviol glycoside mixture with a TSG contentof 98.05% (w/w on a dry basis) comprising 0.18% Rubusoside, 0.78%Dulcoside A, 30.16% Stevioside, 11.25% Rebaudioside C, 2.14%Rebaudioside F, 52.77% Rebaudioside A, 0.59% Rebaudioside D, 0.09%Steviolbioside, and 0.09% Rebaudioside B.

The third stage of purification comprises elution of middle sectioncolumns with an aqueous alcoholic solution in which the water to alcoholratio (vol/vol) is in the range of about 60:40 to 0.1:99.9. The elutionis carried out either from each column separately (parallel connection)or or from two or more consecutively (serially) connected columns.

In one example, the elution of middle section columns at the third stageresulted in a steviol glycoside mixture with a TSG content of 98.56%(w/w on a dry basis) comprising 0.18% Rubusoside, 0.78% Dulcoside A,30.31% Stevioside, 11.31% Rebaudioside C, 2.15% Rebaudioside F, 53.04%Rebaudioside A, 0.59% Rebaudioside D, 0.10% Steviolbioside, and 0.10%Rebaudioside B.

The fourth stage of the process comprises removal of alcohol from thesteviol glycoside eluate obtained after the third stage, and furtherconcentration and drying of the steviol glycoside mixture to obtain adried highly purified mixture of steviol glycosides. Any method known inthe art may be used for ethanol removal, concentration and drying.

Purification of Reb D from a Low Purity Steviol Glycosides Mixture

The present invention is also directed to a method for preparingRebaudioside D, or a purified steviol glycosides mixture andRebaudioside D, including the steps of providing a steviol glycosidesmixture, dissolving the steviol glycosides mixture in a solvent toproduce a solution of steviol glycosides, passing the solution ofsteviol glycosides through a multi-column system including a pluralityof columns packed with an adsorbent resin, eluting impurities from themulti-column system with water or an aqueous solution, eluting highRebaudioside D fractions from the multi-column system with a firstaqueous alcohol solution, crystallizing Rebaudioside D from solutionsprepared from the high Rebaudioside D fractions, eluting steviolglycoside mixture fractions from the multi-column system with a secondaqueous alcohol solution, and drying the steviol glycoside mixturefractions to obtain the purified steviol glycosides mixture.

According to the present invention the purification of steviolglycosides was developed from a steviol glycosides mixture with a TSGcontent of less than 95% w/w on a dry basis.

In one example the TSG content of an initial low purity steviolglycoside mixture was 84.3% w/w on a dry basis, and the Rebaudioside Dcontent was 0.5% w/w on dry basis.

A column system as described above was used to purify a mixture ofsteviol glycosides.

In one embodiment, the method for isolation and purification ofRebaudioside D comprises passing an aqueous or aqueous alcoholicsolution of a low purity steviol glycoside mixture through aconsecutively connected column system. The water to alcohol ratio(vol/vol) in the aqueous alcoholic solution is 99.9:0.1 to 60:40. As aresult different steviol glycosides are retained in different sectionsof the column system.

In one embodiment the columns with retained steviol glycosides aresubjected to elution of compounds with a lower affinity, morespecifically compounds other than steviol glycosides, in order toincrease the purity level of the final products. Lower affinitycompounds are subsequently eluted with an aqueous or aqueous alcoholicsolution of an acid, aqueous or aqueous alcoholic solution of a base,and an aqueous alcoholic solution, in a specific order. The water toalcohol ratio (vol/vol) in the aqueous alcoholic solution is 99.9:0.1 to60:40. The elution of lower affinity compounds is carried out eitherfrom each column separately (parallel connection) or from two or moreconsecutively (serially) connected columns.

A fraction with a high Rebaudioside D content is eluted from columnswith an aqueous alcoholic solution. The water to alcohol ratio (vol/vol)in the aqueous alcoholic solution is in the range of 99.9:0.1 to 50:50.The elution of fractions with a high Rebaudioside D content may becarried out either from each column separately (parallel connection) orfrom two or more consecutively (serially) connected columns.

The remaining steviol glycoside fractions may be eluted from each columnseparately (parallel connection) or from two or more consecutively(serially) connected columns with an aqueous alcoholic solution. Thewater to alcohol ratio (vol/vol) in the aqueous alcoholic solution is inthe range of 60:40 to 0.1:99.9.

The remaining steviol glycoside fractions may be subjected to drying toyield a highly purified steviol glycoside mixture with at least 95%(w/w) dry basis TSG content.

In one example the actual yield of the highly purified steviol glycosidemixture was 72% from theoretical. The combined Rebaudioside D fractionscontained 15.1% Rebaudioside D (w/w) on a dry basis.

Fractions with a high Rebaudioside D content may be combined and mixedwith aqueous alcohol. The water to alcohol ratio (vol/vol) in theresulting aqueous alcoholic solution is in the range of 99.9:0.1 to0.1:99.9. Crystallization of Rebaudioside D is induced by addingRebaudioside D crystals and maintaining the mixture at 0-60° C. withagitation during a period of from about 30 min to about 7 days. Crystalsof Rebaudioside D are separated to yield a product with at least 60%(w/w) Rebaudioside D content on a dry basis.

In another embodiment high Rebaudioside D fractions are combined anddried, and then mixed with aqueous alcohol. The water to alcohol ratio(vol/vol) in the resulting aqueous alcoholic solution is in the range of99.9:0.1 to 0.1:99.9. Crystallization of Rebaudioside D is induced byadding Rebaudioside D crystals and maintaining the mixture at 0-60° C.with agitation during a period of from about 30 min to about 7 days.Crystals of Rebaudioside D are separated to yield a product with atleast 60% (w/w) Rebaudioside D content on a dry basis.

In one example the first crystallization yielded a product with at least86% (w/w) Rebaudioside D content on a dry basis.

In another embodiment Rebaudioside D is further purified by suspendingand agitating in water or aqueous alcohol at 0-60° C. during a period offrom about 30 min to about 7 days, and then separating the RebaudiosideD crystals to obtain Rebaudioside D with a 95% (w/w) dry basis purity.The water to alcohol ratio (vol/vol) in the applied aqueous alcoholicsolution is 99.9:0.1 to 0.1:99.9. If necessary this step may be repeatedseveral times to obtain highly purified Rebaudioside D with at least 95%(w/w) dry basis purity.

In one example the yield of highly purified Rebaudioside D with 98.2%(w/w) dry basis purity was 70% of the Rebaudioside D content in theinitial low purity steviol glycoside mixture.

Purification of Reb D from Plant Biomass

Diterpene glycosides, including sweet-tasting substances, are found indifferent parts of the S. rebaudiana Bertoni plant, being present at thehighest concentration in the leaves. The leaves, therefore, are thepreferred starting material for recovery of sweet glycosides.

The process of the present invention provides for complete retreatmentof Stevia rebaudiana Bertoni plant extract, with isolation andpurification of a highly purified steviol glycoside mixture or highlypurified individual sweet glycosides, such as Rebaudioside D. The plantextract can be obtained using any method such as, but not limited to,the extraction methods described in U.S. Pat. No. 7,862,845, the entirecontents of which are incorporated by reference herein.

One method for purifying Rebaudioside D from a Stevia rebaudiana Bertoniplant biomass, in accordance with the present invention, includes thesteps of providing the biomass of the Stevia rebaudiana Bertoni plant,producing a crude extract by contacting the biomass with water,separating insoluble material from the crude extract to obtain a firstfiltrate containing steviol glycosides, and treating the first filtrateto remove high molecular weight compounds and insoluble particles,thereby obtaining a second filtrate containing steviol glycosides. Thesecond filtrate is then treated with an ion-exchange resin to removesalts, thereby obtaining a resin-treated filtrate. The method furtherincludes passing the resin-treated filtrate through a multi-columnsystem including a plurality of columns packed with an adsorbent resin,to obtain at least one column having adsorbed steviol glycosides;treating the at least one column having adsorbed steviol glycosides witha washing solution to remove impurities; eluting the adsorbed steviolglycosides from the at least one column having adsorbed steviolglycosides, to obtain an eluted solution; treating the eluted solutionto obtain a decolorized solution; evaporating solvent from thedecolorized solution to obtain a high Rebaudioside D content mixture;combining the high Rebaudioside D content mixture with solvent to form aRebaudioside D solution or suspension; and crystallizing Rebaudioside Dfrom the Rebaudioside D solution or suspension to obtain Rebaudioside Dcrystals.

In one embodiment of the present invention, Rebaudioside D was isolatedand purified at the extraction stage as follows. The dried and groundbiomass of Stevia rebaudiana Bertoni was extracted by water, andremaining solids were separated by filtration. The filtrate wassubjected to deionization and decolorization. Then, the resultingfiltrate was passed through a series of chromatographic columns toseparate high and low Rebaudioside D fractions. The resulting solutionswere deionized, decolorized, concentrated and dried. Highly purifiedRebaudioside D was obtained from the concentrate by treating withalcohol or alcohol-water solutions at various temperatures and times.Low Rebaudioside D fractions were combined, evaporated, deionized,decolorized, concentrated, and dried to produce a mixture of steviolglycosides with not less than 95% purity.

According to one aspect of the invention, a method for producingpurified Reb D comprises the steps of: providing a biomass of Steviarebaudiana Bertoni; producing a crude extract by contacting the biomassof Stevia rebaudiana Bertoni with an extracting solvent, such as water;separating insoluble material from the first extract to obtain afiltrate containing steviol glycosides; deionizing the filtrate; passingthe filtrate feed over a series of columns packed with polar resin andeluting steviol glycosides to obtain eluates containing high Reb D andlow Reb D fractions; decolorizing the solutions; evaporating anddeionizing; concentrating by nano-filters and drying.

The process comprises drying of plant material at temperatures betweenabout 20-60° C. for a period of about 2-3 hours to a moisture content ofabout 5-8% before extraction. The drying temperatures between 40-45° C.are more preferable because under those conditions the sweet glycosidesare not decomposed. The dried plant material is milled. A particle sizebetween about 10-20 mm is preferred. The plant material is thenextracted by water in a continuous reflux extractor. The proportion ofextraction water preferably is from about 20 liters to about 25 litersto one kilogram of leaves. The pH of water can be between about pH 2.0and 7.0, however between pH 2.0 and 3.0 is preferable. A larger amountof water may be used; however, it is not preferable from a practicalstandpoint. The extraction temperature can be in the limits of about25-90° C.; however, the temperatures between 50-60° C. are morepreferable for the neutral pH of the extraction solvent. The extractioncan be carried out in batch conditions; however, a continuous refluxextractor is more preferable as the extraction and separation of theresidue is carried out simultaneously. Moreover, the volume of extractis less and the steviol glycoside content is higher in the case of acontinuous reflux extractor. For the batch process the duration ofextraction may range from about 0.5 hours to 24 hours, preferably from 1hour to 6 hours. The contact time for a continuous process is in therange of about 2.5-3.0 hours.

At the extraction stage the pH of water is critical. In a crude Steviaextract there are many kinds of pigments and it is difficult tocharacterize the decolorization capacity for each pigmentquantitatively. The visible absorption spectrum of the transparentsolution of crude extracts is usually tested by a spectrophotometer.There are strong absorption peaks at 420 and 670 nm. The absorption peakat 420 nm results from red and yellow pigments, and the absorption at670 nm results from chlorophyll. The concentration of chlorophyll in thetransparent solution of crude extracts is very low due to its lowsolubility in water. However, the absorption at the wavelength of 370 nmis stronger than that at 420 nm. Thus, the content of pigments in theproduct is often characterized by measuring E₃₇₀, which is theabsorption at the wavelength of 370 nm for a 1.0% aqueous productsolution with 1.0 cm of thickness.

HPLC analysis was performed on an “Agilent 1100 series” (USA) liquidchromatograph equipped with a four channel solvent delivery system,autosampler, thermostatted column compartment and UV detector (210 nm)interfaced with “Chemstation” data acquisition software. Separation ofsteviol glycosides was performed on a “Zorbax NH₂ 150×4.6 mm; 5 μm”chromatographic column. The flow rate was 1.0 mL/min and the mobilephase was an 80:20 (vol/vol.) acetonitrile and water (containing 0.025%acetic acid) mixture. The reference materials for steviol glycosidequantitation were Reb A, Reb C, Stevioside, and Dulcoside A standardsproduced by Chromadex Inc. (USA). Reb B, Reb D (also available fromChromadex), Reb E, Reb F, Rubusoside and Steviolbioside referencematerials were prepared and characterized at the PureCircle R&D center.The steviol glycosides were identified by their retention times. Theconcentrations were calculated by an external standards method andreported on a dry weight basis.

It was shown that at the lower pH at 20° C. the amount of coloredcompounds is lower while the quantity of extracted steviol glycosidesremain at the same level. The effect of pH on steviol glycosides andcolor extraction is summarized in TABLE 2.

TABLE 2 Total extracted steviol pH glycosides, mg/g of leaves E₃₇₀ ofthe filtrate 2.0 10.4 3.4 5.0 10.2 4.2 7.0 10.0 6.5 9.0 9.8 7.9

Thus, an acidic pH is preferable for the preparation of extract with alow content of pigments. For the acidification of extracting water, anymineral or organic acid can be used. However, citric and phosphoricacids are preferable because they can be removed during furthertreatment by calcium oxide.

The variation of extraction rates of individual glycosides at differentpH values and temperatures is summarized in TABLE 3.

TABLE 3 Steviol glycosides, % Conditions St* RebA RebC RebD RebB RebERebF StBio DulA pH 2.0  4° C. 23.6 60.7 11.7 1.7 0.1 0.2 1.2 0.4 0.4 22°C. 21.1 62.0 12.1 1.8 0.1 0.3 1.4 0.7 0.5 50° C. 18.0 64.1 12.2 1.9 0.10.5 1.6 1.0 0.6 pH 5.0  4° C. 20.2 61.1 13.4 2.3 0.1 0.3 2.0 0.1 0.5 22°C. 20.8 61.8 11.5 2.4 0.1 0.4 2.3 0.1 0.6 50° C. 21.0 62.5 9.8 2.5 0.10.5 2.4 0.1 1.1 pH 7.0  4° C. 21.1 63.0 10.1 2.4 0.1 0.5 2.2 0.1 0.5 22°C. 21.0 63.1 9.5 2.6 0.1 0.6 2.4 0.1 0.6 50° C. 20.1 62.6 9.6 2.8 0.10.6 3.0 0.1 1.1 pH 9.0  4° C. 24.6 58.7 10.2 2.9 0.6 0.1 1.9 0.3 0.7 22°C. 25.5 55.3 11.1 3.2 0.8 0.1 2.3 0.5 1.2 50° C. 29.6 48.8 10.2 3.9 1.10.2 4.0 0.8 1.4 *St—Stevioside; StBio—Steviolbioside; DulA—Dulcoside A;Rub—Rubusoside.

Within the pH range of 2.0-7.0, the extraction rate of Steviosideremains practically the same; however, at alkaline pH values itincreases with increasing temperature. The extraction rate of Reb Agenerally increases with the increase of temperature and remainspractically the same at the pH range of 2.0-7.0, while at alkaline pHvalues it is significantly lower. At alkaline pH values the content ofminor glycosides such as Reb B, D, and F, Steviolbioside, and DulcosideA are considerably higher as well. While not intending to be bound bytheory, it is likely that at alkaline conditions the transformation ofReb A to Reb B and conversion of Stevioside to the Steviolbioside alsotakes place. The transformation of Stevioside to the Steviolbioside mayoccur at acidic pH as well. Extraction rates of Reb D, E, and F increaseat higher temperatures.

Thus, the application of acidic pH values and moderate temperatures ispreferable with respect to a high content of Reb A, and low contents ofReb B, Reb D and Reb F; however the quantity of Steviolbioside is higherin these conditions. In the case of acidic pH values the extractiontemperature has to be as low as possible to prevent the formation ofSteviolbioside. In the case of alkaline pH values the content ofcoloring compounds is higher. However, if the extract will be used forfurther production of highly purified individual glycosides such as RebA and especially Reb D, the moderate temperatures and pH values around5.0-7.0 are more preferable. In that case the content of Reb B andSteviolbioside in the final extract is low.

The ratio of leaves and water is also important. The higher the ratio,the higher the extracted amount of non-glycosidic compounds, while atthe very low ratios the quantity of solvent is insufficient for thecomplete extraction of sweet glycosides. At continuous conditions and35-40° C. the preferable ratio of leaves to water is within the limitsof about 1:20 to 1:25, wt/vol. (pH 4.5) (TABLE 4). The contact time isaround 150-160 min. An increase in process duration leads to higherlevels of undesirable compounds in the extract, while a short time isnot enough for complete extraction of sweet glycosides (TABLE 5).

TABLE 4 Leaves:Water Total steviol glycosides, ratio mg/g leaves E₃₇₀ ofthe filtrate 1:3.0 50.1 7.1 1:5.0 51.3 5.9 1:7.0 68.2 5.3 1:10.0 74.44.4 1:15.0 85.6 3.8 1:20.0 87.2 3.5 1:25.0 88.4 3.6 1:30.0 89.0 4.11.35.0 93.5 4.4

TABLE 5 Total steviol glycosides, Time, min mg/g leaves E₃₇₀ of thefiltrate 50 67.2 3.0 100 81.4 3.0 150 88.4 3.5 180 89.1 3.9 200 91.2 4.3

Extraction can be done either at batch conditions or by continuouscountercurrent technique. Alternatively, any other types of extractiontechniques can be applied for the primary isolation of Stevia sweetglycosides, such as ultrasonic extraction, pressurized extraction,microwave conditions or combinations thereof.

The plant material is separated from the solution by filtration and thepH of the filtrate is adjusted to about 8.5-10.0 with calcium oxide orhydroxide (about 1.0% from the volume of filtrate) with slow continuousagitation for about 10-15 min. A lower amount of calcium oxide is notsufficient for satisfactory decolorization of the extract, while highconcentrations will require a larger capacity of ion-exchange resins andmore activated carbon in further stages. The concentrations between1.0-2.0% are preferable. The process is preferably carried out in a fastmanner, as high pH promotes the formation of Reb B, which further willimpact the efficiency of the purification of individual glycosides andthe taste profile of the extract.

The pH of the suspension obtained after treatment with calcium oxide isadjusted to pH 3.0-4.0, preferably to pH 3.0-3.5, by adding FeCl₃. Othersoluble trivalent ferrous salts may be used. The mixture is maintainedfor 10-15 min with slow agitation. The process is preferably carried outin a fast manner, as low pH promotes the conversion of Stevioside toSteviolbioside, which further will affect the efficiency of thepurification of individual glycosides and the taste profile of theextract.

A small amount of calcium oxide is further added to adjust the pH toaround 8.5-9.0, and the mixture is maintained for about 15-40 min,preferably 25-30 min, with slow agitation.

This scheme of chemical treatment results in an extract of slightlyyellowish color. The effect of chemical treatment on the decolorizationof extract is summarized in TABLE 6.

TABLE 6 Stage pH E₃₇₀ of the filtrate After CaO-I  8.5-10.0 2.5 AfterFeCl₃ 3.0-4.0 1.6 After CaO-II 8.5-9.0 0.8

The separation of precipitate can be carried out in any type offiltration system.

The filtrate was subjected to the preliminary deionization by treatmentwith a cation-exchange resin such as Amberlite FPC22H (H⁺) followed withan anion-exchange resin such as Amberlite FPA53 (OH⁻) in a conventionalmanner in continuous conditions. Generally any type of strongcation-exchanger and weak anion-exchangers can be used in this stage.The flow rate in the case of both resins was maintained between bedvolumes (BV) of 0.5-1.0 hour⁻¹. The anion-exchanger shows significantdecolorizing ability. After this treatment the E₃₇₀ value of thefiltrate decreases up to 0.12-0.15. Upon completion of the mixture, theion-exchange resin columns were washed with RO water to recover thesteviol glycosides left in the columns. The deionization stage isimportant for the decolorizing of product, for more efficient furtheradsorption of steviol glycosides on specific resins, and for preventionof adsorbent resin damage.

The deionized solution was passed through consecutively connectedcolumns packed with specific polar macroporous polymeric adsorbent. Theadsorption rate of some impurities such as sterebins is significantlyhigher than that for steviol glycosides. On the other hand duringdesorption stage the impurities desorb faster than sweet compounds,which results in a low quality of final extract. In particular, thesteviol glycosides content is only about 85-86% and the E₃₇₀ is around0.13. To solve this problem and increase the efficiency of this stage,an additional column with specific adsorbent was applied in the firstposition as an “impurities catcher”. The size of the “catcher column”was about ⅓ of the others. The BV was around 1.0 hour⁻¹. Upon completepassage of solution, the resin sequentially was washed with 1 volume ofwater, 2 volumes of 0.5% NaOH, 1 volume of water, 2 volumes of 0.5% HCl,and finally with 2 volumes of water until it reached a neutral pH.

Desorption of the adsorbed steviol glycosides was carried out with50-52% ethanol at BV=1.0-1.5 hour⁻¹. Desorption of the first “catchercolumn” was carried out separately and its eluate was separated from themain solution obtained from other columns. The E₃₇₀ of the filtrateobtained from the “catcher column” was about 0.8, while for the othercolumns it was around 0.035-0.04. Steviol glycoside content in the“catcher column” was significantly low as well. The quality of extractfrom different columns with specific macroporous adsorbent is shown inTABLE 7.

TABLE 7 Columns Total steviol glycosides, % E₃₇₀ of the filtrate“Catcher” 62.3 0.80 First 82.4 0.041 Second 87.6 0.040 Third 92.8 0.040Fourth 93.2 0.039 Fifth 93.4 0.039 Sixth 93.4 0.039

During the adsorption process the column outlets were monitored andsamples were taken from first glycoside containing fractions (firstsweet fractions) of each column and analyzed. In a seven column system,where the first one is a “catcher”, it was shown that if the quantity ofthe steviol glycosides passed through the columns was around 9.5-9.7%from total volume of the columns, the content of Stevioside decreasedfrom 34.9% in the outlet of the “catcher” column to 9.0% in the sixth.At the same time the content of Reb A increased from 44.8% of initialextract to 73.0% in the first portion of the sixth column. Similarly thecontent of Reb D increased from 2.1% of initial extract to 12.0% in thefirst portion of the last column. Apparently, the adsorption rate of RebD is lower compared with Stevioside and Reb A. In the case of Reb C andDulcoside A the reverse correlation is revealed. The ratio of variousglycosides at the outlet of each column (first sweet fraction) duringadsorption, in the case of 120 g of total solids subjected to thetreatment, is summarized in TABLE 8.

TABLE 8 Steviol glycosides, % Column St RebA RebC RebD RebB RebE RebFStBio DulA “Catcher” 34.9 45.2 13.9 2.3 1.1 0.7 0.9 0.1 0.9 First 26.352.9 13.3 3.8 1.0 0.8 1.0 0.1 0.8 Second 22.6 57.5 11.4 4.8 1.2 0.7 1.10.1 0.6 Third 14.8 65.0 8.3 6.9 2.3 1.0 1.4 0.1 0.2 Fourth 13.1 67.6 8.16.6 2.1 0.8 1.4 0.1 0.2 Fifth 11.1 68.7 7.3 7.7 2.1 1.3 1.5 0.1 0.2Sixth 9.0 73.0 3.6 12.0 0.1 0.9 1.2 0.1 0.1 Initial 35.1 44.8 13.3 2.10.3 0.5 2.3 0.1 1.5 extract

The content and ratio of various glycosides in the subsequent portions(after first sweet fraction) of column effluent changed significantly.Reb A, D, and B contents decreased with the volume of column effluent,while the quantity of Reb C and Stevioside increased. The contents ofother glycosides were practically at the same level. However aftersaturation of the resin in all columns the composition of effluent wasalmost the same as that for the initial extract.

The content of various glycosides in various fractions of columneffluent during adsorption is shown in TABLE 9.

Thus, Reb A, D, B, and F contents were increasing before resinsaturation, but decreasing again after saturation of the resin.Stevioside and Reb C contents were decreasing during initial stages ofadsorption, and increasing again with the volume of effluent. Nosignificant changes were observed in the content of other glycosides.Therefore, to produce an extract with a high content of Reb A or lowcontent of Stevioside the initial portions of solution at the outlet ofthe columns has to be collected separately. To increase the quantity ofthat type of product additional sections of the columns can be used. Toproduce Stevia extract with a low content of Reb D the solution has tobe collected after saturation of the resin, i.e. the first sweetfractions of the column effluent have to be excluded. It is notable thatthe Reb D content in column effluent changes rapidly. The Stevia extractwith a low content of Reb C can be produced if collecting anintermediate portion of column effluent. The Reb B quantity can bemaintained at a low level, if the extraction and purification process iscontrolled.

TABLE 9 Effluent:Resin ratio, Steviol glycosides, % vol/vol. St RebARebC RebD RebB RebE RebF StBio DulA 1:1.2 9.0 73.0 3.6 12.0 0.1 0.9 1.20.1 0.1 1:1.3 12.2 71.0 6.1 6.2 2.2 0.9 1.2 0.1 0.1 1:1.4 16.0 67.7 7.95.0 1.3 0.8 1.1 0.1 0.1 1:1.6 19.0 64.8 8.6 4.3 1.1 0.7 1.2 0.1 0.21:2.0 25.1 58.5 10.2 3.3 0.7 0.6 1.2 0 0.4 1:2.3 27.0 56.7 10.6 2.9 0.60.6 1.1 0 0.5 1:2.5 28.6 55.1 10.9 2.8 0.3 0.5 1.2 0 0.6 1:3.0 29.1 54.011.5 2.7 0.5 0.4 1.2 0 0.6 1:3.5 31.8 50.4 12.1 2.6 0.5 0.7 1.1 0 0.81:4.0 33.5 48.7 12.6 2.2 0.4 0.4 1.1 0 1.1 1:4.5 34.4 47.7 13.4 2.2 0.50.5 1.2 0 0.1 1:5.0 35.0 46.9 13.6 2.2 0.5 0.5 1.2 0 0.1 Initial 35.144.8 13.3 2.1 0.3 0.5 2.3 0.1 1.5 extract

A similar correlation was observed in the case of applying 7.5-8.0% ofsolids to the total volume of resin. The content of Stevioside among theadsorbed glycosides was higher in the initial columns and decreasing inthe following ones while the content of Reb A increased from the firstcolumn to the sixth column. The affinity of Stevioside to the adsorbentis significantly higher than that of Reb A. The Reb C content adsorbedon the resin was practically unchanged in the first four columns anddecreased significantly from the fifth column. The Reb D contentadsorbed on the resin is significantly lower than in the columneffluent; however, it is increasing in the final sections. The contentsof Reb E, Reb F, and Dulcoside A decrease from the first to finalsection, as shown in TABLE 10, in the case of 90 g of total solidssubjected to the treatment using six columns, with 200 mL resin in eachcolumn. Thus, if the elution of each column is carried out separately,the Stevia extract with a high content of Stevioside, Reb A or Reb D anddesired amounts of other glycosides can be produced.

TABLE 10 Steviol glycosides, % Column St RebA RebC RebD RebB RebE RebFStBio DulA Initial 35.1 44.8 13.3 2.1 0.3 0.5 2.3 0.1 1.5 Extract First40.7 39.1 11.1 1.5 0.2 0.5 2.4 0.1 4.4 Second 38.1 40.6 12 1.8 0.4 0.52.5 0 4.1 Third 36.4 42.1 12.6 2.5 0.6 0.4 2.7 0 2.7 Fourth 31.7 50 11.22.8 0.8 0.2 2.5 0 0.8 Fifth 20.1 63.7 8.1 4.2 1.8 0.2 1.5 0 0.4 Sixth10.3 66.6 4.4 15.4 2.0 0.2 1.0 0 0.1

However, when the adsorbent is saturated with glycosides the ratio ofvarious glycosides become practically the same in all the columns.

The decrease in the quantity of Stevia extract subjected to thechromatographic treatment results in an increase of Reb D content in thefinal column. However the quantity has to be enough for more than fivecolumns in the case of a six column system. The correlation betweenStevia extract quantity and Reb D content in the final column issummarized in TABLE 11, in the case of six columns which are each packedwith 200 mL resin.

TABLE 11 Quantity of Stevia extract, g RebD content in the sixth column,% 116.0 4.1 110.0 7.3 95.0 13.2 90.0 15.4 85.0 35.3

A flow-chart of the isolation of the high Reb D and low Reb D containingfractions, during the extract purification stage on macroporous polarresin, is presented in FIG. 4.

FIG. 5 illustrates the isolation of high Reb D fractions from Steviaextract adsorbed on macroporous resin.

In the case when the steviol glycoside quantity in the feed exceeds theadsorption capacity of the resin, the steviol glycosides unable toadsorb due to saturation of the resin will “escape” through the systemand form a water fraction. This water fraction is distinguished by ahigh content of Reb D and Reb A and low content of Stevioside. Thecontent of steviol glycosides in different columns and water fractions,in the case of 130 g of Stevia extract subjected to the treatment usingsix columns packed with 200 mL of macroporous resin, is summarized inTABLE 12. The contents of Reb A and Reb D in the water fraction aredecreasing with the increase of the quantity of feed extract. Thecontent of steviol glycosides in the water fraction at variousquantities of initial extract subjected to the treatment, using sixcolumns which are each packed with 200 mL of macroporous resin, issummarized in TABLE 13.

TABLE 12 Steviol glycosides, % Column Stevioside RebA RebC RebD InitialExtract 33.7 53.2 11.3 1.8 First 40.9 45.9 12.3 0.9 Second 41.7 46.111.6 0.6 Third 40.3 48 10.7 1.0 Fourth 37.7 50.1 11.0 1.2 Fifth 35.152.9 10.7 1.3 Sixth 28.5 60.7 8.8 2.0 Water fraction 7.4 70.4 3.7 18.5

As mentioned above the adsorbed steviol glycosides are eluted during thedesorption stage. Higher or lower concentrations of ethanol can be usedfor desorption. However, at concentrations lower than 50% desorptiontime increases significantly, while higher concentrations result inlower quality of the extract.

TABLE 13 Quantity of Stevia Steviol glycosides, % extract, g SteviosideRebC RebA RebD Initial extract 33.7 11.3 53.2 1.8 120.0 3.5 4.2 71.520.8 130.0 7.4 3.8 70.4 18.4 140.0 20.5 9.8 60.1 9.6

The combined solution of steviol glycosides eluted from macroporousadsorbent was treated with activated carbon. High Reb D fractions werecollected separately. The solution from the “catcher column” was notmixed with others. The high Reb A and Stevioside fractions may also becollected separately. The quantity of the carbon was in the range of0.1-0.8% (wt/vol.), and preferably 0.25-0.3% (wt/vol.). The suspensionwith continuous agitation was maintained at 20-25° C. for 20-120 min,and preferably for 25-35 min. Separation of used carbon was conducted onthe plate-and-frame press filter; however, filtration can be performedby any other equipment suitable to separate fine particles fromsolution. This treatment is very efficient for decolorizing, and fordecreasing the E₃₇₀ value of the steviol glycosides up to 0.03absorbance units. Application of larger quantities of activated carbonis not desirable because of strong adsorption of steviol glycosides tothe carbon and thus loss of the product.

The further purification and decolorization of the solution wasaccomplished by treatment with ion-exchange resins. For that purpose theethanol solution of steviol glycosides, after treatment with activatedcarbon, was passed through columns packed with cation-exchange resinAmberlite FPC22H (H⁺) followed with anion-exchange resin Amberlite FPA53(OH⁻). Generally any type of strong cation-exchanger and weakanion-exchangers can be used at this stage. The flow rate for bothresins was between BV=0.5-1.0 hour⁻¹. This treatment has a significantdecolorizing effect, decreasing the E₃₇₀ value of the filtrate up to0.02 absorbance units. To wash out coloring substances adsorbed on theion-exchangers, the columns were washed with 2% NaOH solution in 60% ofethanol.

After passing through the ion-exchange columns, the solution wascollected and transferred to a distillation device. The recovery ofethanol may be carried out on any type of evaporator, with or withoutvacuum. The solids content in the final concentrate was about 13-15%.

The evaporated concentrate was subjected to additional treatment with acation-exchange resin such as Amberlite FPC22H (H⁻) followed with ananion-exchange resin such as Amberlite FPA53 (OH⁻) in a conventionalmanner in continuous conditions. The flow rate in both columns wasmaintained between BV=0.5-1.0 hour⁻¹. After completely passing thesolution through the columns, both resins were washed with tap water torecover the steviol glycosides left in the columns. After this treatmentthe E₃₇₀ of the filtrate was around 0.01-0.014 absorbance units.

The refined extract was transferred to the nano-filtration device andconcentrated to about 52% of solids content. Alternatively theconcentration can be carried out in any evaporating device, preferablyunder reduced pressure conditions. However, nano-filtration ispreferable, because it allows the removal of residual amounts oflow-molecular weight impurities. Moreover, the process is carried out atambient temperature and mild conditions. When vacuum evaporators ordistillation devices are used there is a possibility of Reb B andSteviolbioside formation at elevated temperatures.

The purified extract was spray dried in a conventional manner.

Steviol glycoside content in an extract purified in this manner was inthe range of 96-97%, and the E₃₇₀ of a 1% solution was about 0.012absorbance units. The specific rotation was in the range of −30.5 to−36.6° depending on the Reb A and Stevioside content in the extract.Heavy metals content was less than 1 ppm and ash content was less than0.1%.

The combined fractions with a high content of Reb D eluted from thespecific resin were decolorized, deionized and concentrated as describedabove.

The content of Reb D in the solutions can be increased up to about70-80%, after chromatographic re-treatment similar to that describedabove. Further purification of Reb D can be carried out either fromconcentrated solution or powder.

A flow chart illustrating the isolation of high Reb D containingfractions in the extraction stage, from the portion of Stevia extractnot adsorbed on macroporous resin, is presented in FIG. 6.

To carry out the purification of Reb D from concentrated solution, thesolution was mixed with anhydrous methanol and stirred at 20-22° C. for24 hours. The best output was obtained at a 33-35% solids concentration.The effect of the solids concentration of the initial syrup, subjectedto precipitation using anhydrous methanol in the ratio of 1:2(vol/vol.), on the yield of crude Reb D is summarized in TABLE 14.

TABLE 14 Concentration, % Reb D yield, % from maximum 25.0 21 30.0 3633.0 100 35.0 100 37.0 95 40.0 62 50.0 23 60.0 12

The best ratio of Reb D concentrate to methanol was 1:2 (vol/vol.). Atthe lower ratios no precipitation took place, as shown in TABLE 15.

TABLE 15 RebD Concentrate/Methanol Ratio Reb D yield, % from maximum1:0.5 0 1:1.0 0 1:1.5 57 1:2.0 100 1:2.5 81 1:3.0 64 1:3.5 53 1:4.0 44

The purity of Reb D was in the range of 84-88%.

To effect further purification the crystals were suspended in a mixtureof alcohol and water; agitated at 40-75° C., preferably 55-60° C., for5-60 min, preferably 15-30 min; cooled down to 20-22° C.; and agitatedfor another 1-5 hours, preferably 1-2 hours.

At this stage both ethanol and methanol may be used. Water content isessential to produce high purity product. The effect of the solvent'swater content when the ratio of crude Reb D to methanol-water solutionis 1:3 (wt/vol.) is summarized in TABLE 16.

TABLE 16 Concentration of methanol RebD purity, % Yield, % Crude RebD84.3 — 99.0 85.6 98.4 95.0 86.1 98.2 90.0 87.2 97.9 85.0 87.8 97.6 80.088.6 97.5 75.0 89.3 97.2 70.0 89.6 96.8 65.0 92.4 95.1 60.0 95.8 91.755.0 95.8 88.3 50.0 95.1 73.3 45.0 95.1 71.6 40.0 94.9 69.3

When ethanol was used, the best results were obtained at the 45-55%concentration. However, when using ethanol the mixing time is preferablyabout 12-18 hours to get a high degree of purification.

At this stage the purification may also be carried out by washing withwater.

To produce Reb D with 99% or greater purity, the step of washing with analcohol-water solution or water may be repeated.

The purification of crude Reb D in powder form was carried out bycrystallization from an alcohol-water solution or washing with water, asdescribed above.

The purification of crude Reb D in powder or syrup form from the lastcolumn fraction of the specific resin was carried out according to theschemes described above.

Purification of Reb D Alone with Reb A from Stevia Extract

In another embodiment of this invention Reb D purification is carriedout using commercial Stevia extract as a starting material. The contentof Reb D in the extract can vary depending on the Stevia plant varietyor the technological scheme of the extract preparation.

In another embodiment of the present invention, the isolation andpurification of Rebaudioside D and Rebaudioside A was carried usingStevia extract as a starting material. The method comprises thetreatment of Stevia extract with alcohol or alcohol-water solutions toisolate Rebaudioside A with a relatively high content of Rebaudioside D;separation of the crystals and their treatment with alcohol oralcohol-water solutions to produce highly purified Rebaudioside A inprecipitate, and a filtrate with high Rebaudioside D content; drying theprecipitate to produce high purity Rebaudioside A; evaporation ofalcohol from the filtrate; and treating the concentrated or driedsolution of Rebaudioside D with alcohol or alcohol-water solutions atvarious temperatures and times to produce high purity Rebaudioside D.

Unless indicated otherwise, Stevia extract containing Stevioside at22.63%, Reb A at 52.46%, Reb C at 7.52%, Reb D at 1.18%, Reb B at 0.55%,Reb E at 0.64%, Reb F at 1.79%, Steviolbioside at 0.10%, and Dulcoside Aat 1.59%, with 88.46% total steviol glycosides content, was used asstarting material in this embodiment.

In one embodiment of the present invention, Stevia extract was dissolvedin an ethanol-water solution at 50-70° C., preferably 55-60° C.;incubated for about 10-30 min, preferably 15-20 min; and then at 15-40°C., preferably 20-22° C., for about 18-48 hours, preferably 20-24 hours,with agitation. When the temperature reached 22° C., 1-2% (wt/vol.) ofhighly purity (>97%) Reb A was added to the reaction mixture as astarter to initiate crystallization. The proportion of extract andethanol-water solution varied depending on the content of minorcompounds, particularly Reb B, D and Steviolbioside, and was between1.0:2.5 and 1.0:10.0 (wt/vol.), and preferably between 1.0:3.0 and1.0:5.0 (wt/vol.).

The concentration of ethanol may be from 75-99%, and preferably from82-88%.

During incubation precipitate was formed, which was separated byfiltration or centrifugation. The purity and yield of Reb A depended onthe extract to ethanol ratio and concentration of ethanol (TABLES 17 and18). The content of Reb A and Reb D in the final product ranged between80-99% and 0.8-3.5% respectively. Therefore, this scheme is useful forthe one-stage production of highly purified Reb A.

TABLE 17 Extract:ethanol ratio, Steviol glycosides, % Ethanol, % wt/vol.St RebC RebA RebD Yield, % 75 1:3.0 0.1 0.2 98.9 0.8 19.2 78 -″- 0.1 0.298.6 1.1 21.3 80 -″- 0.1 0.2 98.3 1.4 23.4 82 -″- 0.1 0.2 97.9 1.8 23.785 -″- 0.1 0.2 97.7 2.0 24.1 87 -″- 0.3 0.4 96.8 2.5 25.6 88 -″- 0.4 0.595.6 3.5 33.0 89 -″- 1.0 0.7 94.8 3.5 35.4 90 -″- 1.4 1.2 94.4 3.0 35.795 -″- 3.2 3.1 91.2 2.5 41.6 99 -″- 7.2 10.3 80.4 2.1 48.3

TABLE 18 Extract:ethanol Ethanol, ratio, Yield of RebA % wt/vol.Product, % content, % RebD content, % 88.0 1:5.0 26.5 98.0 1.5 88.01:4.0 31.3 97.5 2.0 88.0 1:3.5 32.4 96.9 2.6 88.0 1:3.0 33.0 95.6 3.588.0 1:2.5 35.6 91.7 2.7 88.0 1:2.0 41.4 89.8 3.2

The Reb D content increases with the increase of ethanol concentrationand decrease of solution to solids ratio. At the same time, the purityof Reb A increases in the case of more diluted ethanol solutions andhigher ratios of ethanol to extract.

The precipitate, separated by filtration or centrifugation, was washedwith about two volumes of absolute ethanol and dried.

The yield of the product (Reb A) at this stage for Stevia extracts withvarious contents of Reb A, after treatment with 88% ethanol (1:3 wt/vol.ratio), is summarized in TABLE 19. As could be expected, the yield ofthe product increases with the increase of the content of Reb A in theinitial extract.

TABLE 19 Yield of Reb A at precipitation stage from Reb A content ininitial extract, % initial extract, % 42.0-43.0 28.0-30.0 45.0-46.032.0-35.0 50.0-53.0 32.0-38.0 55.0-59.0 41.0-42.0 60.0-62.0 45.0-46.0

If the initial extract contains high amounts of Reb B and Reb D, lowerconcentrations of ethanol and a higher ratio of ethanol to the extractare preferred for Reb A, and later Reb D, purification (TABLE 20; TABLE21).

The yield of Reb A and Reb D can be increased by using ethanol forso-called “after-precipitation”. For that purpose, at the end ofcrystallization 0.5-1.0 (vol/wt), preferably 0.5-0.8 (vol/wt), ofabsolute ethanol to the initial solids, was added to the mixture and theprocess was continued for another 2-3 hours. The yield and purity of theproduct from an extract with 48.7% of Reb A content are summarized inTABLE 22.

TABLE 20 Ratio ethanol Purity of product at different Reb B content,Ethanol, to solid, % (Reb D content was 0.4%) % vol/wt 0% 0.4% 0.8% 1.1%81.0 2.5 98.7 98.5 98.2 97.9 3.0 98.9 98.7 98.4 98.1 3.5 99.2 98.9 98.698.4 83.0 2.5 98.1 98.2 98.0 97.7 3.0 98.5 98.4 98.2 97.9 3.5 98.8 98.698.4 98.2 85.0 2.5 97.7 97.6 97.4 97.2 3.0 98.2 97.9 97.6 97.4 3.5 98.598.2 97.8 97.6 87.0 2.5 96.3 97.2 96.6 96.4 3.0 97.5 97.6 97.4 97.0 3.597.9 97.9 97.6 97.2 88.0 2.5 96.1 95.9 95.5 95.1 3.0 97.3 97.1 96.4 95.83.5 97.7 97.5 97.2 96.8 90.0 2.5 94.6 94.1 92.3 90.5 3.0 96.3 95.8 92.891.2 3.5 97.3 96.8 93.7 91.9

TABLE 21 Ratio ethanol Purity of product at different content of Reb D,Ethanol, to solid, % (Reb B content was 0.1%) % vol/wt 0.5% 1.2% 1.7%2.6% 81.0 2.5 98.7 98.0 97.5 97.1 3.0 98.9 98.3 98.0 97.4 3.5 99.2 98.598.2 97.7 83.0 2.5 98.1 97.7 97.3 97.0 3.0 98.5 98.1 97.8 97.4 3.5 98.898.4 98.0 97.6 85.0 2.5 97.7 97.5 97.1 96.8 3.0 98.2 97.8 97.6 97.1 3.598.5 98.1 97.8 97.2 87.0 2.5 96.3 96.2 95.8 94.2 3.0 97.5 97.3 96.7 96.13.5 97.9 97.6 97.4 96.9 88.0 2.5 96.1 95.7 95.2 93.7 3.0 97.3 97.1 96.595.6 3.5 97.7 97.4 97.0 96.6 90.0 2.5 94.6 94.2 93.5 93.0 3.0 94.8 94.893.9 93.3 3.5 95.7 95.4 94.4 93.5

TABLE 22 Additional ethanol Yield and purity of RebA at differentconcentrations of ethanol volume, (ratio of ethanol to extract = 1:3.5,wt/vol.) vol/wt to 85% 86% 87% 88% solids Yield, % RebA, % Yield, %RebA, % Yield, % RebA, % Yield, % RebA, % 0 29.5 98.5 30.6 98.3 32.797.9 33.3 97.8 0.5 31.4 98.5 31.6 98.2 33.4 97.9 33.8 97.6 0.6 32.3 98.232.7 98.2 34.3 97.8 34.7 97.6 0.7 33.5 97.9 33.9 97.7 35.4 97.6 35.997.5 0.8 34.1 97.9 35.2 97.7 36.3 97.6 36.7 97.4 0.9 34.3 97.8 35.4 97.636.7 97.5 37.4 97.4 1.0 34.5 97.8 35.7 97.5 36.9 97.4 37.7 97.2

To produce high purity Reb A the process can be carried out at 30-50° C.without a cooling stage. However, when this was done, although thepurity of Reb A was higher the process resulted in a lower yield of theproduct. The quality of the product increased at higher washingtemperatures. The results obtained using 3.5 volumes of 85% ethanol toone part of extract after 24 hours, and with and without anafter-precipitation stage, are summarized in TABLE 23.

TABLE 23 Yield, % Content of Reb A With after- With after- Withoutprecipitation Without precipitation Temperature, after- (0.8 vol. after-(0.8 vol. ° C. precipitation EtOH) precipitation EtOH) 22.0 29.6 33.598.2 98.5 30.0 28.7 32.8 98.4 98.6 35.0 27.5 32.2 98.7 98.9 40.0 27.031.4 98.8 99.2 45.0 25.4 28.9 99.0 99.4 50.0 24.3 25.6 99.2 99.5 Reb A,Reb B and Reb D contents in extract were 51.3, 0.2% and 0.7%,respectively.

When the content of Reb A in the final product was less than 97% mainlydue to high content of Reb B and/or Reb D, the product was additionallywashed with an aqueous solution of ethanol. The Reb A obtained afterprecipitation was suspended in the ethanol-water mixture at roomtemperature for 30-40 min. After homogeneous suspension was obtained thetemperature was increased up to 35-50° C., preferably 38-42° C., andagitated for about 10-20 hours, preferably 12-15 hours, and then at10-25° C., preferably 20-22° C., for about 3-20 hours, preferably 5-10hours. The proportion of Reb A and ethanol was 1.0:2.0-1.0:5.0(wt/vol.), preferably 1.0:2.5-4.0 (wt/vol.). The ethanol concentrationwas between 85-93%, and preferably 88-90%.

If the purity of Reb A was lower than 97% due to a high content ofStevioside, the product was washed with absolute ethanol in the samemanner as described above for Reb B and Reb D contaminated product. Theproportion of Reb A and ethanol was 1.0:2.0-1.0:5.0 (wt/vol.), andpreferably 1.0:2.5-4.0 (wt/vol.)

FIG. 7 illustrates a one-stage purification scheme of Reb A usingethanol-water systems.

The purification of Reb D from the Reb A crystals with a relatively highcontent of Reb D was carried out using various schemes.

According to the one scheme, the material with a relatively high contentof Reb D was mixed with an ethanol-water solution and incubated at45-65° C., preferably 50-55° C., for 2-6 hours, preferably 3-4 hours,with agitation. Then, the mixture was cooled down to room temperaturefor 1-3 hours, preferably 0.5-1 hour. The precipitate with a highcontent of Reb D was separated by filtration or centrifugation.

The preferable ratio of solids to aqueous ethanol solution was 1:5,(wt/vol.), and the optimum concentration of ethanol was 78% (TABLE 24).

TABLE 24 Ratio ethanol to Purity of RebD in Ethanol, % solid, vol/wtRebA, % crystals, % 75.0 4.0 98.5 18.4 5.0 99.2 18.6 6.0 99.4 18.6 77.04.0 98.4 18.7 5.0 99.1 20.1 6.0 99.2 20.3 78.0 4.0 98.4 19.2 5.0 99.222.0 6.0 99.4 22.1 79.0 4.0 98.1 19.0 5.0 98.8 19.7 6.0 99.0 19.8 80.04.0 98.0 17.3 5.0 98.4 17.9 6.0 98.9 18.2 82.0 4.0 97.7 15.2 5.0 98.115.8 6.0 98.7 16.4

The process can be completed in a shorter time period when an aqueoussolution of ethanol is used, however even pure ethanol may be efficient.

In another embodiment an initial material containing 95.6% of Reb A and3.5% Reb D (FIG. 8a ) was mixed with 3.5 volumes of 78.0% ethanol, themixture was boiled for 10-15 min and undissolved crystals were separatedby hot filtration. The output of the crystals was in the range of6.0-8.0%, with 52-53.0% and 43-45.0% of Reb A and Reb D (FIG. 8b )contents, respectively.

For the further purification the precipitate was suspended in 50%ethanol at the ratio of 1:2 (wt/vol.) and at 30-40° C., preferably33-37° C., and maintained for 2-15 hours, preferably 10-12 hours, withagitation. The suspension was filtered and dried. The yield ofprecipitate with about 20-22.0% of Reb A and 80-82% of Reb D was in therange of 34-36.0%. Up to five volumes of aqueous ethanol can be appliedat this stage. The concentration of ethanol can be in the range of10-80%, preferably 45-50%.

The precipitate can be subjected to similar treatment to obtain aproduct with less than 5.0% of Reb A and about 95% of Reb D content(FIG. 8c ). The yield of the product was around 58-60%.

According to another scheme which is presented schematically in FIG. 9,the Reb A crystals with a relatively high Reb D content, obtained afterprecipitation with aqueous ethanol solution, were mixed with methanol oraqueous methanol solution and the mixture was maintained at 20-40° C.,preferably 20-25° C., for 0.5-6 hours, preferably 1-2 hours. The ratioof methanol solution or pure methanol to solids was in the range of1.5-10, preferably 1:2 (wt/vol.) to 1:3 (wt/vol.). At a higher ratio theresulting solution contains a higher level of Reb A. In all of theexamples the purity of Reb A in the “washed” crystals was not less than98% (TABLE 25).

Generally ambient temperature is sufficient to wash out Reb D; however,higher temperatures may also be used.

The precipitate was separated by filtration or centrifugation, washedwith about two volumes of anhydrous methanol, and dried.

TABLE 25 Ratio methanol to Purity of RebA RebD in filtrate, Methanol, %solid, vol/wt crystals, % % dry basis 100.0 2.0 98.3 24.5 3.0 98.9 28.35.0 99.3 22.2 80.0 2.0 98.5 19.7 3.0 98.9 20.6 5.0 99.3 18.6 70.0 2.098.5 19.7 3.0 99.1 21.5 5.0 99.4 20.2 60.0 2.0 98.7 25.5 3.0 99.3 28.65.0 99.5 22.3 50.0 2.0 98.7 25.9 3.0 99.4 29.3 5.0 99.5 22.4 40.0 2.098.7 21.6 3.0 99.4 24.5 5.0 99.5 20.4 30.0 2.0 98.8 21.9 3.0 99.5 24.85.0 99.6 20.8 20.0 2.0 98.4 20.3 3.0 98.5 21.5 5.0 98.5 20.1

The purity of Reb A was 98.3-99.6%.

The remaining solution and washed liquid were combined and methanol wasremoved by distillation.

For the further purification of Reb D the filtrate was concentrated andmixed with anhydrous methanol and the resulting mixture was intensivelyagitated for at least 24 hours. For the precipitation of Reb D thepreferable concentration of solids in initial solution was in the rangeof 33-37% (TABLE 26).

TABLE 26 Concentration of Ratio of solids to Purity of Remainingsolution, % methanol, wt/vol. RebD, % 20.0 1:2.0 42.4 25.0 1:2.0 47.330.0 1:2.0 55.4 33.0 1:2.0 84.4 35.0 1:2.0 84.3 37.0 1:2.0 83.6 40.01:2.0 75.1 45.0 1:2.0 No precipitate 50.0 1:2.0 No precipitate

The ratio of solid to liquid may vary in the range of 1:2.0-1:7.0(wt/vol.), preferably 1:2.0-3.5 (wt/vol.). However at a higher ratio theoutput of precipitate decreases (TABLE 27).

TABLE 27 Concentration of Ratio of solids to Purity of Remainingsolution, % methanol, wt/vol. RebD, % 33.5 1:1.5 73.5 33.5 1:2.0 84.533.5 1:2.5 84.7 33.5 1:3.0 84.7 33.5 1:3.5 83.5 33.5 1:4.0 75.7 33.51:5.0 71.6 33.5 1:6.0 64.3 33.5 1:7.0 61.1

The precipitate was separated by filtration or centrifugation, washedwith about two volumes of anhydrous methanol and dried.

In order to obtain higher purity Reb D the precipitate was suspended ina methanol-water mixture. A concentration of methanol between 30-80% canbe used, preferably 55-60%, which results in a higher purity and yieldof Reb D. The suspension was heated up to 40-65° C., preferably 50-55°C., and maintained for 10-15 minutes, cooled down to 20-22° C. andmaintained for an additional 1.0 to 4.0 hours, preferably 1.0-1.5 hours,with agitation. The preferred ratio of solid to methanol solution wasfrom 1:3.0 to 1:5.0 (wt/vol.), more preferably 1:3.0 (TABLE 28).

TABLE 28 Solids to methanol Purity of Methanol, % ratio, wt/vol. RebD, %30.0 1:2.0 98.5 1:3.0 98.7 1:4.0 98.6 1:5.0 98.3 40.0 1:2.0 97.6 1:3.098.5 1:4.0 98.5 1:5.0 98.2 50 1:2.0 98.6 1:3.0 98.9 1:4.0 98.8 1:5.098.5 60 1:2.0 98.8 1:3.0 99.1 1:4.0 99.1 1:5.0 98.3 70 1:2.0 98.4 1:3.098.6 1:4.0 98.5 1:5.0 97.5

The suspension was filtered and dried as described above.

The purity of Reb D was around 98-99% at the optimum conditions.

Alternatively the remaining solution was concentrated to 60-70% ofsolids, and after adding 1% (from total solids) Reb D crystals as astarter, it was allowed to incubate for 48-72 hours. The resulting masswas then mixed with 2-3 volumes of anhydrous methanol, and then thesuspension was stirred at ambient temperature for 2-3 hours.

The crystals were separated and dried as described above. The purity ofReb D was about 84-85%.

The further purification of the material was carried out according tothe procedure described above using a 55-60% methanol solution.

Purification of Reb D from Remaining Solution after Isolation of Reb A

In another embodiment of the present invention the isolation andpurification of Rebaudioside D was developed from solutions remainingafter the isolation of Rebaudioside A from Stevia extract. RebaudiosideA purification was carried out by a one-, two- or three-stage process,by treatment with various alcohols or alcohol-water systems at varioustemperatures and times. The isolation of a high Rebaudioside D fractionfrom remaining solutions was developed using chromatographic separationtechniques at various conditions. From crude preparations, RebaudiosideD was purified by treatment with alcohol or alcohol-water solutions atvarious temperatures and times. Low Rebaudioside D fractions werecombined, evaporated, deionized, decolorized, concentrated, and dried toproduce a mixture of steviol glycosides with not less than 95% purity.

Commercial Stevia extract containing Stevioside at 24.3%, Reb A at62.1%, Reb C at 8.8%, Reb D at 1.6%, Reb B at 0.2%, Reb E at 0.6%, Reb Fat 1.0%, Steviolbioside at 0.10%, and Dulcoside A at 1.3%, with 87.9% oftotal steviol glycosides content, was used as a starting material inmost of the experiments for this embodiment.

Stevia extract was dissolved in a methanol-water solution at 10-70° C.,preferably 55-60° C., for about 10-30 min, preferably 15-20 min, andthen at 15-40° C., preferably 20-22° C., for about 10-15 minutes, andmaintained for 2-4 hours, preferably 1-2 hours, with agitation. When thetemperature reached 20-22° C., 1-2% of highly purified Reb A was addedto the reaction mixture as a starter to initiate crystallization. Theproportion of extract and methanol-water solution depended on thecontent of Reb A and other compounds including minor glycosides, and wasbetween 1.0:2.5-1.0:10.0 (wt/vol.), preferably 1.0:3.0-5.0, (wt/vol.).

The concentration of methanol may be between 75-99%, preferably 82-88%.The content of Reb A and Reb D in the final product ranges from 97% to99% and 0.2-0.4% respectively at optimum and near to optimum conditions.Therefore this embodiment is useful for the one-stage production ofhighly purified Reb A.

The data are summarized in TABLES 29 and 30.

TABLE 29 Extract:methanol Steviol glycosides, % Methanol, % ratio,wt/vol. St RebC RebA RebD Yield, % 75 1:3.0 0.2 0.5 99.2 0.1 14.4 78 -″-0.3 0.5 99.1 0.1 19.6 80 -″- 0.4 0.6 98.8 0.2 21.2 82 -″- 0.5 0.7 98.60.2 32.2 85 -″- 0.6 0.6 98.5 0.3 40.3 87 -″- 0.5 0.8 98.4 0.3 42.5 88-″- 0.8 1.0 97.9 0.3 44.4 89 -″- 1.0 1.0 97.7 0.3 45.6 90 -″- 1.1 1.297.3 0.4 48.1 95 -″- 1.8 1.6 96.2 0.4 48.6 99 -″- 3.2 4.7 91.4 0.7 49.3

TABLE 30 Extract:methanol ratio, Steviol glycosides, % Methanol, %wt/vol. St RebC RebA RebD Yield, % 85 1:5.0 0.1 0.4 99.4 0.1 40.5 851:4.0 0.2 0.5 99.2 0.1 41.8 85 1:3.5 0.4 0.6 98.8 0.2 42.3 85 1:3.0 0.60.6 98.5 0.3 42.5 85 1:2.5 0.8 1.3 97.5 0.4 44.3 85 1:2.0 3.6 4.3 91.30.8 47.7

Under optimum conditions the major quantity of Reb D remains in thefiltrate. In crystals the quantity of Reb D is increasing with adecrease of methanol solution to solids ratio. At the same time thepurity of Reb A is increasing in cases of more diluted methanolsolutions and higher ratios of methanol to extract.

The yield of Reb A from Stevia extract of various qualities aftertreatment with 85% methanol at 1:3 wt/vol. ratio is summarized in TABLE31.

TABLE 31 RebA content in initial extract, % Yield of RebA, % 42.0-43.019.0-23.0 45.0-46.0 25.0-28.0 50.0-54.0 28.0-31.0 55.0-59.0 33.0-37.060.0-62.0 38.0-43.0

The precipitate was separated by filtration or centrifugation, washedwith about two volumes of absolute methanol and dried.

The yield of crystals may be increased using methanol forafter-precipitation. For that purpose at the end of crystallization,0.5-1.0% (vol/wt), preferably 0.7-0.8% (vol/wt), of absolute methanol tothe initial solids, was added to the reaction mixture and the processwas continued for another 20-30 minutes.

The yield and purity of the crystals and remaining solution for theextract with 62.1% Reb A and 1.6% Reb D content are summarized in TABLE32.

TABLE 32 Additional methanol Extract:methanol volume, ratio, vol/wt toSteviol glycosides, % Methanol, % wt/vol. solids St RebC RebA RebDYield, % 85.0 1:3.0 0 0.6 0.6 98.5 0.3 42.5 85.0 1:3.0 0.2 0.6 0.7 98.40.3 43.4 85.0 1:3.0 0.5 0.6 1.0 98.1 0.3 44.3 85.0 1:3.0 0.7 0.6 1.298.0 0.2 46.9 85.0 1:3.0 0.8 0.5 1.4 98.0 0.1 47.4 85.0 1:3.0 0.9 0.51.6 97.7 0.2 47.5 85.0 1:3.0 1.0 0.5 1.9 97.4 0.2 47.5

When the content of Reb A in the crystals was less than 97%, theprecipitate was washed with additional volumes of anhydrous methanol.

The process of purification of Reb A using methanol can be carried outin continuous conditions in fully automated systems.

The one-stage purification scheme of Reb A using methanol as a solesolvent, and isolation of Reb D from the remaining solution, ispresented in FIG. 10.

In another embodiment of the invention, a Reb A purification scheme wassimilarly developed using ethanol as a solvent.

The purification of Reb D from the remaining solution was developedusing various schemes.

The remaining solution, after isolation, of Reb A using ethanol ormethanol solutions, was evaporated and passed through a series of eightconsecutively connected columns with the rate equal to 1.0 bed volumeper 1 hour. Each column was packed with 200 mL of specific polarmacroporous polymeric resin. Upon completion of the adsorption the resinwas washed with 5 volumes of water.

Desorption of the adsorbed steviol glycosides was carried out separatelyfrom each column with 50-52% ethanol at BV=1.0-2.0 hour⁻¹.

Similarly to Stevia extract in the case of applying 9.5-10.0% of totalsolids to the total volume of resin, the content of Stevioside and Reb Cis higher in the initial columns and decreases in the following ones,while the content of Reb A and Reb D increases from the first column tothe eighth column. The data shows that affinity of Stevioside to theadsorbent is significantly higher than that of Reb A. The contents ofReb E, Reb F, and Dulcoside A decrease from the first to the finalcolumn. The steviol glycoside composition adsorbed on each column underconditions described above is presented in TABLE 33.

Thus, if the elution of each column is carried out separately a steviolglycoside mixture with a high content of Stevioside, Reb A, or Reb D andcontrolled amounts of other glycosides can be obtained.

TABLE 33 Steviol glycosides, % Quantity, Column St RebA RebC RebD RebBRebE RebF StBio DulA g Initial 51.9 21.6 20.5 2.8 0.4 0.4 1.2 0.1 1.1152.0 mixture First 53.3 16.5 24.7 0.8 0.4 0.7 1.8 0.3 1.5 18.6 Second53.6 17.0 24.0 0.8 0.4 0.7 1.8 0.2 1.5 17.2 Third 53.3 21.0 21.3 0.4 0.50.5 1.7 0.1 1.2 16.2 Fourth 51.8 23.0 20.5 1.0 0.5 0.4 1.5 0.1 1.2 14.3Fifth 48.5 26.3 20.2 1.5 0.5 0.4 1.1 0.1 1.4 12.1 Sixth 39.8 34.7 19.62.7 0.5 0.3 1.0 0.0 1.4 12.3 Seventh 32.6 40.1 18.1 6.7 0.6 0.2 1.0 0.00.7 11.5 Eighth 20.9 48.5 9.0 19.6 0.4 0.2 0.8 0.0 0.6 7.8 Yield, % 70.798.0 79.2 90.4 94.5 89.4 92.6 87.9 88.9 72.3

However, when the adsorbent is saturated the ratio of various glycosidesbecomes practically the same in all columns.

The decrease in total solids subjected to the chromatographic treatmentresults in an increase of Reb D content in the final column. However thequantity has to be enough for steviol glycosides to reach the finalcolumn. The correlation between glycosides quantity subjected to thechromatographic separation and Reb D content in the final eighth columnis shown in TABLE 34. Each column was packed with 200 mL of macroporousresin.

TABLE 34 Quantity of Glycosides, g Reb D content in the eight column, %160.0 6.7 152.0 19.6 150.0 21.3 145.0 25.8

Similarly to Stevia extract, in the case of higher quantities whichexceed the adsorption capacity of the resins, the resulting “waterfraction” contains high amounts of Reb D and Reb A and lower amounts ofStevioside. The yield of total product was in the range of 73-75%. Reb Dand Reb A recovery was around 90%. The content of steviol glycosides indifferent columns and water fractions is shown in TABLE 35.

TABLE 35 Steviol glycosides, % Quantity, Column St RebA RebC RebD RebBRebE RebF StBio DulA g Initial 51.9 21.6 20.5 2.8 0.4 0.4 1.2 0.1 1.1170.0 mixture First 51.0 18.9 24.5 0.9 0.4 0.7 1.8 0.3 1.5 18.2 Second51.2 20.0 23.6 0.6 0.4 0.7 1.8 0.2 1.5 17.5 Third 51.2 23.3 21.0 0.4 0.60.5 1.7 0.1 1.2 15.4 Fourth 50.4 24.4 21.0 0.4 0.6 0.4 1.5 0.1 1.2 14.5Fifth 50.0 25.2 20.8 0.7 0.5 0.4 1.1 0.1 1.2 14.4 Sixth 49.0 25.5 20.41.5 0.5 0.3 1.1 0.1 1.6 12.3 Seventh 47.8 26.8 20.1 2.3 0.2 0.2 1.0 0.11.5 12.4 Eighth 36.1 31.8 18.6 11.2 0.2 0.1 1.0 0.0 1.0 11.5 Water 17.536.8 16.2 27.6 0.5 0.6 0.7 0.0 0.5 7.7 fraction Yield, % 72.0 90.9 81.590.3 86.5 86.0 90.0 96.8 93.5 72.9

The content of Reb A and Reb D in the “water fraction” decreases withthe increase of total solids subjected to the purification, as shown inTABLE 36 for an eight column system, wherein each column is packed with200 mL macroporous resin.

TABLE 36 Steviol glycosides, % Total solid, g St RebC RebA RebD OthersInitial solution 51.9 20.5 21.6 2.8 3.2 180.0 30.7 19.6 31.9 13.3 4.5170.0 17.5 16.2 36.8 27.6 1.9 167.0 17.3 13.9 38.5 28.7 1.6 164.0 17.013.2 39.2 29.3 1.3

The content of Reb D in the solutions increased up to 70-80% afterchromatographic re-treatment, which is described above.

The desorption of steviol glycosides, and further treatment andpurification of Reb D and other glycosides, was carried out in a similarmanner as that described above for the Stevia extract.

Flow charts of chromatographic isolation of high Reb D fractions fromthe remaining solution after isolation of Reb A using methanol treatmentare presented in FIGS. 11 and 12.

The purity of Reb D was about 98-99% at the optimum conditions.

The purity of the mixture of steviol glycosides after isolation of Reb Dwas not less than 95%.

Purification of Reb D from Remaining Solution after Isolation of Reb aand Steviol Glycosides

In another embodiment of the present invention, the isolation andpurification of Rebaudioside D was developed from remaining solutionswhich were obtained after the isolation of Rebaudioside A, and a highlypurified mixture of other steviol glycosides, from a Stevia extract.

Rebaudioside A isolation from a steviol glycoside mixture containing ahigh amount of Rebaudioside D was carried out by a one-, two- orthree-stage process using treatment with various alcohols oralcohol-water systems, at various temperatures and times. The combinedremaining solution was treated with an alcohol solution, separated anddried to produce a highly purified mixture of steviol glycosides. Theisolation of a high Rebaudioside D fraction from the remaining solutionswas developed using chromatographic separation techniques. From thecrude preparations, Rebaudioside D was purified by treatment withalcohol or alcohol-water solutions at various temperatures and times.

Commercial Stevia extract containing Stevioside at 21.4%, Reb A at54.6%, Reb C at 7.7%, Reb D at 1.4%, Reb B at 0.2%, Reb E at 0.5%, Reb Fat 0.9%, Steviolbioside at 0.10%, and Dulcoside A at 1.1%, with 91.2% oftotal steviol glycosides content, was used as starting material in mostof the experiments of this embodiment.

Isolation of Reb A from Stevia extract using ethanol or methanolsolutions was carried out as described above.

The content of steviol glycosides in the remaining solution (motherliquor) was: Stevioside (48.2%), Reb A (14.7%), Reb C (11.1%), Reb D(1.4%), Reb B (0.2%), Reb E (1.2%), Reb F (1.8%), Steviolbioside (0.4%),Dulcoside A (1.9%), and Rubusoside (1.4%). The total steviol glycosidescontent was 82.3%.

In order to produce a highly purified mixture of steviol glycosides thespray-dried mother liquor was dissolved in absolute methyl alcohol andmaintained at 60-75° C., preferably 65-70° C., for about 5-20 min,preferably 5-10 min, and then at 10-35° C., preferably 20-25° C., forabout 6-24 hours, preferably 9-12 hours, with agitation. The proportionof powdered mother liquor and methanol was 1.0:2.5 to 1.0:10.0(wt/vol.), preferably 1.0:3.0 to 1.0:5.0 (wt/vol.). The precipitate wasseparated by filtration or centrifugation.

A higher methanol to solid ratio resulted in a higher purity of steviolglycosides and a higher content of Stevioside. At the lower amounts ofmethanol the yield of steviol glycosides was higher; however, the puritywas lower. In contrast, higher amounts of methanol resulted in a higherpurity and lower yield of the product. Different quantities of methanolresulted in different ratios of steviol glycosides (TABLE 37).

TABLE 37 Solid to methanol, Steviol glycosides, % TSG, Yield, wt/vol. StRebA RebC RebD RebB RebE RebF StBio DulA Rub % % Initial 48.2 14.7 11.11.4 0.2 1.2 1.8 0.4 1.9 1.4 82.3 — mixture 1:2.5 69.3 19.3 4.6 0.5 0.10.6 0.7 0.3 0.4 0.5 96.3 59.5 1:3.0 69.6 21.0 4.6 0.5 0.1 0.6 0.7 0.30.3 0.4 98.1 48.2 1:3.5 72.5 19.1 4.3 0.5 0.1 0.6 0.6 0.3 0.1 0.4 98.546.6 1:4.0 75.8 16.2 3.9 0.5 0.1 0.4 0.6 0.4 0.4 0.6 98.9 45.4 1:4.584.1 11.8 2.2 0.3 0.1 0.1 0.3 0.1 0.1 0.1 99.2 43.8 1:5.0 86.2 10.8 1.70.2 0.1 0.1 0.1 0.1 0 0 99.3 40.9 1:6.0 88.1 10.4 0.4 0.1 0.1 0 0.1 0.10 0 99.3 38.6 1:7.0 93.2 5.6 0.3 0.1 0.1 0 0.1 0.1 0 0 99.5 33.5

The precipitate was separated by filtration or centrifugation, washedwith about two volumes of absolute methanol and dried.

The filtrate and washing liquid were combined and spray dried. Thecontent of steviol glycosides in the filtrate after precipitation with3.0 volumes of methanol was as follows: Reb A (15.6%), Stevioside(4.5%), Reb C (28.9%), Reb B (0.8%), Reb D (4.8%), Reb E (3.3%), Reb F(3.7%), Dulcoside A (4.2%), Steviolbioside (0.5%), and Rubusoside(2.2%). The total content of glycosides was 68.5%.

This stage may also be carried out using anhydrous ethanol or aqueoussolutions of methanol and ethanol.

Purification of Reb D from the remaining solution was carried out bycolumn chromatography using macroporous polar resins as carriers.

The remaining solution was passed through a series of six columns at arate equal to 1.0 bed volume per 1 hour. Each column was packed with 200mL of specific polar macroporous polymeric resin. Upon completion of theadsorption the resin was washed with 5 volumes of water.

In order to obtain higher quantity and quality of glycosides the numberof the columns may be increased.

Desorption of the adsorbed steviol glycosides was carried out separatelyfor each column with 50-52% ethanol at BV=1.0-2.0 hour⁻¹.

When applying around 9.5-10.0% of total solids to the total volume ofresin, all the glycosides were adsorbed on the resin. The contents ofStevioside and Reb C increased slightly in the first columns anddecreased in later ones. The concentration of Reb A, Reb E, Reb F andReb D increased gradually from the first column to the last one.Dulcoside A decreased from the first to final column. The purity of themixture of glycosides was higher in the last columns.

The data obtained when passing the remaining solution containing 115 gof solids through the six columns, each packed with 200 mL ofmacroporous resin, are summarized in TABLE 38.

Reb D content in the last column increased to around 31%.

Similarly to the remaining solution obtained after isolation of Reb A,the decrease of the amount of total solids subjected to thechromatographic treatment results in an increase of Reb D content in thefinal column.

TABLE 38 Steviol glycosides, % Purity, Column St RebA RebC RebD RebBRebE RebF StBio Rub DulA % Initial 4.5 15.6 28.9 4.8 0.8 3.3 3.7 0.5 2.24.2 68.5 mixture First 5.7 16.5 32.2 0.3 0.5 0.5 0.6 0.1 2.7 4.0 63.7Second 7.2 20.8 33.9 0.3 0.5 0.5 1.9 0.2 2.3 3.2 70.8 Third 7.9 24.632.1 0.4 0.5 0.5 2.5 0.2 1.8 1.5 72.0 Fourth 7.2 29.8 30.6 0.5 0.5 0.52.8 0.1 1.2 1.1 74.3 Fifth 6.5 42.4 25.3 12.4 0.6 1.9 1.1 0.1 0.1 0.290.6 Sixth 2.2 45.9 6.7 31.3 0.7 5.4 4.9 0 0 0 97.1

The correlation between total solids quantity subjected to theseparation on the six columns, each with 200 mL volume, and Reb Dcontent in the final column is summarized in TABLE 39.

TABLE 39 Total solids, g Reb D content in the sixth column, % 117.0 23.6115.0 31.3 110.0 39.4 107.0 42.9

In the case of higher quantities which exceed the adsorption capacity ofthe resins, the resulting “water fraction” contains a high amount of RebD and Reb A and a lower content of Stevioside.

The content of steviol glycosides in different columns and the waterfraction using 125 g of initial material is presented in TABLE 40.

The contents of Reb A and Reb D in the water fraction decrease with theincrease in quantity of the total solids subjected to the purification.The content of steviol glycosides in the water fraction at variousquantities of the initial mixture, when six columns, each packed with200 mL of resin, were used is presented in TABLE 41.

TABLE 40 Steviol glycosides, % TSG, Column St RebA RebC RebD RebB RebERebF StBio Rub DulA % Initial 4.5 15.6 28.9 4.8 0.8 3.3 3.7 0.5 2.2 4.268.5 mixture First 4.3 15.0 31.8 0.9 0.4 0.3 0.6 0.1 2.3 4.1 59.8 Second4.2 25.7 31.6 0.9 0.4 0.3 0.7 0.2 2.0 2.8 68.8 Third 4.0 30.4 28.6 1.20.5 0.5 0.9 0.2 1.5 1.3 69.1 Fourth 3.1 36.9 28.2 1.6 0.5 0.7 1.2 0.11.1 1.1 74.5 Fifth 2.8 42.3 26.9 2.5 0.6 1.7 1.3 0.1 0.1 0.2 78.5 Sixth1.3 46.5 25.7 6.3 0.7 2.1 2.4 0 0 0 85.0 Water 0.9 18.4 12.4 26.3 0.94.8 4.7 0 0 0 68.4 fraction

The content of Reb D in the solutions increased up to 70-80% alterchromatographic re-treatment, which is described above. PP-60

TABLE 41 Steviol glycosides, % Total solid, g St RebC RebA RebD OthersInitial mixture 4.5 28.9 15.6 4.8 17.7 121.0 0.9 11.9 19.3 31.1 6.1125.0 0.9 12.4 18.4 26.3 10.4 128.0 1.4 12.9 18.1 22.2 11.9 130.0 1.613.3 17.6 20.8 12.4

The desorption of steviol glycosides, and further treatment andpurification of Reb D and other glycosides, was developed similarly tothe methodology described above for the Stevia extract and remainingsolution obtained after isolation of Reb A.

The purity of Reb D was around 98-99% at the optimum conditions.

The purity of the mixture of steviol glycosides after isolation of Reb Dby a second chromatographic treatment using the same macroporous resinwas not less than 95%.

Remaining solution for the purification of Reb D can be obtained bydifferent schemes for the purification of Reb A and complex retreatmentof Stevia extract. Depending on the purity of the initial extract, Reb Aand minor compounds, especially Reb D and Reb B contents, the processcan be carried out by a three-, two- or one-stage process. Reb Dpurification schemes after Reb A isolation by a one-stage processutilizing ethanol or methanol was described above. Reb D purificationschemes after Reb A isolation by a three- and two-stage process isdescribed below.

Description of the Purification of Reb D after Isolation of Reb A by aThree-Stage Process

When Reb A content in the initial extract is low or Reb D and/or Reb Bcontents are high the three-stage process is preferred in order toprepare highly purified Reb A. In principle, by this process Reb A canbe purified from almost any type of Stevia extract. For that purposeStevia extract was dissolved in absolute ethyl alcohol and maintained at70-90° C., preferably 80-85° C., for about 5-20 min, preferably 5-10min, and then at 10-25° C., preferably 18-22° C., for about 6-24 hours,preferably 9-12 hours, with agitation. The proportion of extract andethanol was 1.0:2.5 to 1.0:10.0, wt/vol., and preferably 1.0:3.0 to1.0:5.0, wt/vol. The precipitate was separated by filtration orcentrifugation.

Stevia extracts containing Stevioside (9.0-52.0%), Reb A (29.1-74.7%),Reb C (3.6-13.3%), Reb D (2.1-12.0%), Reb B (0.1-2.3%), Reb E(0.5-1.3%), Reb F (0.9-2.3%), Steviolbioside (0-0.1%), and Dulcoside A(0.1-1.5%), with 86-95% of total steviol glycosides content were used asstarting material.

The higher the content of Reb A in the initial extract, the greater theamount of ethanol which is used at this stage. Higher ethanol:extractratios result in higher purity of Reb A. If the amount of ethanol wasdecreased, the yield of crude Reb A was higher, but the purity waslower. In contrast, a higher amount of ethanol resulted in Reb A ofhigher purity but lower yield.

For this stage at least 98% ethanol is preferred. However, anethanol-water solution can be used in a one- or two-stage purificationprocess of Reb A, which will be discussed below.

If the extract contains more than 55% Reb A, the precipitation occurswithout heating of the mixture but results in a low quality product.

Precipitation may also take place from extracts with lower than 40% RebA content; however, the yield and quality of the final product is lowand the process takes up to 20-24 hours.

The content of Reb A in the final product ranges between 75-85% and theyield depends on the Reb A content in the initial extract (TABLE 42) andthe extract:ethanol ratio (TABLE 43).

TABLE 42 Yield of crude RebA content in Extract:Ethanol RebA, % frominitial extract, % ratio, wt/vol. initial extract RebA content, %42.0-43.0 1:3.0 48.0-50.0 75.0-76.0 45.0-46.0 ″ 50.0-55.0 76.0-77.050.0-53.0 ″ 55.0-60.0 77.0-79.0 55.0-59.0 ″ 71.0-72.0 80.0-81.060.0-62.0 ″ 75.0-76.0 81.0-83.0

The precipitate was separated by filtration or centrifugation and washedwith about one volume of absolute ethanol. The filtrate and washedliquid were combined and spray dried.

The typical compositions of the crystals and mother liquor in this stageare summarized in TABLE 44.

For further purification, the precipitate with about 75-83% of Reb Acontent was completely dissolved in the ethanol-water mixture by heatingat 80-85° C. for 30-40 min with continuous agitation.

TABLE 43 Yield of crude RebA content in Extract:Ethanol RebA, % frominitial extract, % ratio, wt/vol. initial extract RebA content, % 52.61:2.0 58.2 72.1 ″ 1:2.5 57.6 75.4 ″ 1:3.0 56.4 79.8 ″ 1:3.5 54.3 80.1 ″1:4.0 53.1 81.2 ″ 1:4.5 52.7 82.3 ″ 1:5.0 52.0 82.6 ″ 1:6.0 49.8 82.9 ″1:7.0 47.4 83.5 ″ 1:8.0 44.1 83.9 ″ 1:9.0 38.2 84.5 ″  1:10.0 34.4 85.1

TABLE 44 Steviol glycosides, % Product St RebA RebC RebD RebB RebE RebFStBio DulA Crystals  5-10.0 75.0-83.0 5-10.0 0.2-2.5 <1.0 <1.0   5-10.0<0.5 <0.5 Mother 55-67.0  14-19.0 9-15.0 1.5-3.5 0-1.5 0.2-1.5 0.2-4.00-0.4 0.7-2.0 liquor

The obtained solution was cooled to 20-25° C. and pure Reb A was addedin the amount of 1-2% from solids. The suspension was cooled further toa temperature of 14-15° C., and maintained for 12-14 hours with slowagitation. Then, 0.5-1.5, vol/wt, preferably 0.7-1.0, vol/wt, ofabsolute ethanol to the initial solids was added to the crystallizationmixture. Reb A continued to crystallize for another 12-14 hours.

The precipitate was separated by filtration or centrifugation and washedwith about one volume of absolute ethanol. The purity of Reb A in thisstage was 95-96% with a yield of 75-77%.

The filtrate and wash liquid were combined, spray dried and used for thepurification of Reb D as described above for remaining solutions.

The purity of Reb A increased at lower concentrations and larger amountsof ethanol.

The process is preferably carried out without drying of intermediatecrude Reb A, as the solubility of the product and purificationefficiency decreases significantly after drying. In the case of driedmaterial, ethanol with lower concentrations must be applied, whichresults in lower yields of product. The concentration of ethanol can bebetween 70-90%, preferably 75-85%. The ratio of solid to liquid may varyin the range of 1:2.0-1:5.0, wt/vol., preferably 1:2.2-3.5, wt/vol. Athigher concentrations of ethanol, use of a higher ratio is preferred andvice versa. The optimum concentration of ethanol in this stage was 80.5%and the best ratio of solid to ethanol solution was 1:2.3, wt/vol.(TABLE 45; TABLE 46).

TABLE 45 Ethanol Crude RebA:ethanol Yield of concentration, % ratio,wt/vol. Product, % RebA content, % 70.0 1:3 32-34 99.2 71.0 ″ 32-34 99.072.0 ″ 33-35 98.8 73.0 ″ 36-37 98.5 74.0 ″ 44-46 98.2 75.0 ″ 50-53 97.976.0 ″ 52-54 97.5 77.0 ″ 56-59 97.3 78.0 ″ 60-62 97.2 79.0 ″ 63-66 96.980.0 ″ 69-70 96.7 80.5 ″ 71-72 96.4 81.0 ″ 73-74 96.1 81.5 ″ 75-76 96.182.0 ″ 76-78 96.0 83.0 ″ 76-79 96.0 85.0 ″ 81-82 95.8 86.0 ″ 82-83 95.687.0 ″ 82-85 95.6 88.0 ″ 84-89 95.5 89.0 ″ 85-89 95.2 90.0 ″ 87-90 95.091.0 ″ 91-92 93.1

TABLE 46 Ethanol Crude RebA:ethanol Yield of concentration, % ratio,wt/vol. RebA, % RebA content, % 80.5 1:5.0 50-55 96.9 80.5 1:4.0 59-6096.6 80.5 1:3.5 67-68 96.5 80.5 1:3.0 71-72 96.4 80.5  1:2.65 72-73 96.480.5 1:2.5 73-74 96.4 80.5 1:2.4 74-75 96.4 80.5 1:2.3 75-77 96.3 80.51:2.2 77-78 94.7 80.5 1:2.0 77-78 94.5

The typical compositions of the crystals and mother liquor in this stageare summarized in TABLE 47.

TABLE 47 Steviol glycosides, % Product St RebA RebC RebD RebB RebE RebFStBio DulA Crystals <1.5 >95.0 <2.0 <1.5 <0.5 <0.5 <1.0 <0.2 <0.3 Mother18-30.0 45-55.0 18-25.0 0.5-4.0 0-1.5 0.2-1.0 1.0-7.0 0-0.5 0.2-3.0liquorPreparation of high purity Reb D was carried out similarly to the othertypes of remaining solutions.

Reb A with purity of 95-96% obtained at the second stage of purificationwas subjected to the re-crystallization in order to obtain a productwith purity not less than 97%. The Reb A was completely dissolved in theethanol-water mixture by heating at 80-85° C. for 30-40 min withcontinuous agitation. To increase the dissolution rate in some cases theReb A may be dissolved in the solutions with lower concentrations ofethanol and, after complete dissolution, the remaining amount of ethanolcan be added.

The obtained solution was cooled to 20-25° C. and pure Reb A was addedin the amount of 1-2% from solids. The suspension continued to cooluntil 14-15° C., and maintained for 12-14 hours with slow agitation.Then, 0.5-1.5, vol/wt, preferably 0.7-1.0, vol/wt, of absolute ethanolto the initial solids, was added to the mixture, and crystallizationcontinued for another 12-14 hours.

The precipitate was separated by filtration or centrifugation, washedwith about one volume of absolute ethanol and dried. The purity of Reb Ain this stage was in the range of 97.2-99.7% with a yield up to 86%.

The filtrate and wash liquid were combined and spray dried.

The quality of product increased at lower concentrations and largeramounts of ethanol.

It is preferable to carry out the process without drying the initialmaterial, as the solubility of the product and purification efficiencydecrease significantly after drying. In case of dried material, ethanolwith lower concentrations has to be applied, which resulted in loweryields of product. The concentration of ethanol can be between 70-82%,preferably 75-80%. The ratio of solid to liquid may vary in the range of1:2.0-1:5.0, wt/vol., preferably 1:2.5-3.5, wt/vol. At higherconcentrations of ethanol, use of a higher ratio is preferred, and viceversa. The optimum concentration of ethanol in this stage was 77.7% andthe best ratio of solid to ethanol solution was 1:3.0-3.5, wt/vol.(TABLE 48; TABLE 49; TABLE 50).

TABLE 48 Ethanol Crude RebA:ethanol Yield of concentration, % ratio,wt/vol. RebA, % RebA content, % 70.0 1:3 46-47 99.7 71.0 ″ 48-50 99.772.0 ″ 57-58 99.6 73.0 ″ 65-66 99.1 74.0 ″ 70-71 99.0 75.0 ″ 79-80 98.976.0 ″ 79-80 98.8 77.0 ″ 80-81 98.8 78.0 ″ 82-83 98.2 79.0 ″ 82-83 98.080.0 ″ 82-84 97.7 80.5 ″ 84-85 97.4 81.0 ″ 84-85 97.3 81.5 ″ 84-85 97.282.0 ″ 85-86 97.2

TABLE 49 Ethanol Crude RebA:ethanol Yield of concentration, % ratio,wt/vol. RebA, % RebA content, % 76.5 1:3 78-80 98.8 77.0 ″ 80-81 98.877.1 ″ 80-81 98.8 77.2 ″ 80-81 98.8 77.3 ″ 80-81 98.7 77.4 ″ 80-81 98.777.5 ″ 80-81 98.7 77.6 ″ 80-81 98.6 77.7 ″ 82-83 98.5 77.8 ″ 82-83 98.377.9 ″ 82-83 98.2 78.0 ″ 82-83 98.2

Final yields of highly purified Reb A from various types of extracts aresummarized in TABLE 51.

The typical compositions of the final product and mother liquor in thisstage are summarized in TABLE 52.

TABLE 50 Ethanol Crude RebA:ethanol Yield of concentration, % ratio,wt/vol. RebA, % RebA content, % 77.7 1:5.0 71-72 99.6 77.7 1:4.0 74-7599.1 77.7 1:3.5 82-83 98.7 77.7 1:3.0 82-83 98.5 77.7  1:2.65 83-84 97.677.7 1:2.5 85-86 97.4 77.7 1:2.4 85-86 97.4

TABLE 51 Yield of RebA Yield of RebA Yield of RebA at recrystallizationat precipitation at crystallization stage from Recovery of RebA contentin stage from stage from initial extract RebA from initial extract, %initial extract, % initial extract, % (Final yield), % initial extract,% 42.0-43.0 48.0-50.0 37-39 31-32 72-73 45.0-46.0 50.0-55.0 41-43 34-3573-74 50.0-53.0 55.0-60.0 44-45 36-38 74-75 55.0-59.0 71.0-72.0 48-4944-45 76-77 60.0-62.0 75.0-76.0 50-51 46-48 77-78

TABLE 52 Steviol glycosides, % Product St RebA RebC RebD RebB RebE RebFStBio DulA Final <0.2 >97.0 <0.2 <1.0 <0.2 <0.3 <0.3 <0.1 <0.1 productMother 3-8.0 75-87.0 3-8.0 2.0-5.0 0-0.5 0.2-1.5 0.5-2.0 0-0.5 0.2-1.0liquor

The concentrated or spray dried filtrate was used as initial materialfor the purification of Reb D and a highly purified mixture of steviolglycosides, using the same methodology described for the other remainingsolutions.

The three-stage purification scheme of Reb A and isolation of Reb Dfraction from remaining solutions is presented in FIG. 13.

Purification of Reb D after Isolation of Reb a by a Two-Stage Process

The first stage of purification was carried out similarly to thethree-stage process described above.

When 70-75% of ethanol was used in the ratio of 1:2.5-3.0, wt/vol.,during the re-crystallization stage the purity of product exceeded 98%,with about 50-53% yield in optimum cases. The purity of productincreased with the decrease of ethanol concentration and increase ofliquid volume. When the TSG content of initial extract is higher than93% and the purity of crude Reb A after the first stage is 84% or more,the 78-81% of ethanol solution can be used in this stage. The ratio ofcrude Reb A to ethanol was 1:2.5-2.65, wt/vol., and crystallization timewas 28-35 hours. The purity of product was around 98% with about 55-57%yield (TABLE 53).

TABLE 53 Yield of RebA, % RebA content, % Ethanol Crude RebA:ethanolCrude RebA:ethanol concentration, ratio, wt/vol. ratio, wt/vol. % 1:2.51:2.65 1:3.0 1:2.5 1:2.65 1:3.0 70.0 34-36 33-35 31-32 98.8 99.1 99.271.0 38-39 37-38 32-34 98.7 99.0 99.0 72.0 40-42 37-38 35-37 98.6 98.798.8 73.0 43-44 41-42 37-39 98.5 98.4 98.5 74.0 45-46 44-46 42-44 98.198.2 98.2 75.0 52-55 50-53 50-51 97.9 98.1 97.9 76.0 54-56 53-55 51-5397.6 97.7 97.5 77.0 57-61 57-60 53-55 97.2 97.4 97.3 78.0 62-64 61-6356-58 96.9 97.3 97.2 79.0 66-68 64-66 59-61 96.6 97.0 96.9 80.0 73-7570-72 67-69 96.1 96.6 96.7 81.0 76-78 73-74 70-72 95.7 96.2 96.1 82.079-80 76-78 74-75 95.4 95.7 96.0

The total final yield of highly purified Reb A from various types ofextracts, using 75% ethanol solution with 1:2.65, wt/vol. ratio ofsolids to liquid, is summarized in TABLE 54.

TABLE 54 Yield of RebA Yield of RebA at precipitation at crystallizationRecovery of RebA content stage from stage from RebA from in initialinitial initial extract initial extract, % extract, % (Final product), %extract, % 42.0-43.0 48.0-50.0 25-26 57-59 45.0-46.0 50.0-55.0 27-2860-61 50.0-53.0 55.0-60.0 29-31 61-62 55.0-59.0 71.0-72.0 37-38 67-6860.0-62.0 75.0-76.0 39-40 65-66

With this purification scheme, when using the same equipment as used inthe three-stage process, the production capacity can be increasedsubstantially.

The filtrate and washing liquid were combined and spray dried. Theresulting material was highly suitable for the production of Reb D usingchromatographic separation techniques as described above.

The typical compositions of the final product and mother liquor in thisstage are summarized in TABLE 55.

TABLE 55 Steviol glycosides, % Product St RebA RebC RebD RebB RebE RebEStBio DulA Final <0.2 >97.0 <0.2 <1.0 <0.2 <0.3 <0.3 <0.1 <0.1 productMother 18-27.0 40-48.0 18-28.0 1.0-5.0 0-1.0 0.4-1.0 2.0-7.0 0-0.50.2-3.0 liquor

The two-stage purification scheme of Reb A, by crystallization andisolation of a high Reb D fraction, is presented in FIG. 14.

When the combined content of Reb B and Reb D in the initial extract islower than 1.7%, the second stage can be carried out by a “washing”technique, without dissolution and crystallization of the productobtained after the first stage.

The crystals of crude Reb A obtained after precipitation were suspendedin the ethanol-water mixture at room temperature for 30-40 min. After ahomogeneous suspension was obtained the temperature was increased up to35-55° C., preferably 40-45° C., and maintained for about 10-20 hours,preferably 12-15 hours, and then at 10-25° C., preferably 20-22° C., forabout 3-20 hours, preferably 5-10 hours, with continuous agitation.Then, 0.5-1.5, vol/wt, preferably 0.7-1.0, vol/wt, of absolute ethanolto the initial solids, was added to the mixture and the process wascontinued for another 12-14 hours. The proportion of crude Reb A andethanol depended on the content of minor compounds, especially Reb B andReb D, and was between 1.0:2.0-1.0:5.0, wt/vol., preferably 1.0:2.5-3.5,wt/vol. The ethanol concentration was between 75-88%, and preferablybetween 79-85%.

The precipitate was separated by filtration or centrifugation, washedwith about one volume of absolute ethanol, and dried. The purity of RebA was greater than 97.0% with a 50-69% yield. The data obtained on crudeReb A material with 1.76% Reb D and 0.5% Reb B content is summarized inTABLE 56. The process was first carried out at 40° C. and then at 22° C.for 12 hours, adding 0.5 volume of ethanol for after-precipitation ateach step.

TABLE 56 Yield of Product, % RebA content, % Ethanol Crude RebA:ethanolCrude RebA:ethanol concentration, ratio, wt/vol. ratio, wt/vol. % 1:2.51:3.0 1:3.5 1:2.5 1:3.0 1:3.5 75.0 51-52 30-32 18-22 98.5 99.1 99.4 76.053-54 33-35 24-25 98.3 98.9 99.2 77.0 54-55 37-38 30-32 98.3 98.7 99.078.0 56-57 48-49 36-38 98.2 98.6 98.8 79.0 58-60 51-53 39-41 98.1 98.698.7 80.0 59-60 52-53 40-41 98.0 98.4 98.5 81.0 62-64 54-55 42-44 97.898.3 98.4 82.0 66-68 55-57 46-47 97.6 98.1 98.2 83.0 68-69 56-58 47-4897.2 97.5 97.7 85.0 69-71 58-59 49-50 96.6 96.8 97.0 88.0 75-77 64-6654-56 96.3 96.5 96.6 90.0 79-80 67-68 58-60 95.8 96.3 96.5 95.0 81-8273-74 70-72 95.3 95.6 96.1

The ethanol precipitation of extracts with higher contents of Reb B, RebD, Reb E and Reb F results in crude Reb A with higher contents of thesecompounds. Reb F and Reb E content decrease simultaneously with Reb C.The higher the content of Reb B and Reb D the lower the ethanolconcentration should be, and the higher the ethanol to solids ratio. Athigher concentrations of ethanol and a lower ratio of ethanol to solids,the yield of final product was higher but the purity was lower. Lowerconcentrations of ethanol and higher ratios result in a higher purityproduct with lower yield. From materials with low Reb B and Reb Dcontent the yield and quality of product were higher. In this case,higher concentrations of ethanol can be used to increase the final yieldof the product (TABLE 57, TABLE 58).

When the process was carried out without a cooling stage, the purity ofReb A was higher; however, it resulted in a lower yield of the product.The quality of the product increased at higher washing temperatures.Similar results were obtained during the process without anafter-precipitation stage (TABLE 59).

TABLE 57 Ratio ethanol Purity of product at different RebB content,Ethanol, to solid, % (RebD content was 1.1%) % vol/wt 0% 0.2% 0.6% 0.9%79.0 2.5 98.1 98.0 97.9 97.4 3.0 98.6 98.5 98.3 97.5 3.5 98.7 98.5 98.097.7 80.0 2.5 98.0 98.0 97.7 97.2 3.0 98.4 98.4 97.9 97.5 3.5 98.5 98.598.1 97.8 82.0 2.5 97.6 97.6 97.4 97.1 3.0 98.1 98.0 97.6 97.2 3.5 98.298.2 97.9 97.0 85.0 2.5 96.6 96.5 96.2 95.6 3.0 96.8 96.7 96.5 95.9 3.597.0 97.0 96.7 96.3

TABLE 58 Ratio ethanol Purity of product at different content of RebD,Ethanol, to solid, % (RebB content was 0.2%) % vol/wt 0.6% 1.1% 1.5%2.1% 79.0 2.5 98.3 98.0 97.6 97.2 3.0 98.5 98.5 97.8 97.5 3.5 98.8 98.598.3 97.9 80.0 2.5 98.2 98.0 97.4 97.1 3.0 98.4 98.4 97.9 97.3 3.5 98.698.5 98.1 97.6 82.0 2.5 97.8 97.6 97.2 96.9 3.0 98.2 98.0 97.6 97.1 3.598.4 98.2 97.9 97.4 85.0 2.5 96.8 96.5 95.9 94.1 3.0 97.2 96.7 96.4 94.43.5 97.4 97.0 96.6 95.3

Thus, depending on the quality of initial material and goals of theprocess the optimal technological parameters can be varied to achievethe desired result.

The concentrated or spray dried filtrate was used as initial materialfor the purification of Reb D and a highly purified mixture of steviolglycosides using the same methodology as was described for the otherremaining solutions.

The two-stage purification scheme of Reb A, using a “washing” techniqueand isolation of high Reb D fraction, is presented in FIG. 15.

TABLE 59 Additional ethanol volume, Quality of product washed with 82%ethanol with ratio of 1:2.5, wt/vol. vol/wt to 22° C. 35° C. 40° C. 45°C. 50° C. 55° C. solids A* B** A B A B A B A B A B 0 61.6 98.1 58.2 98.657.4 98.8 55.3 98.9 45.1 99.4 32.5 99.6 0.5 65.7 97.7 60.5 98.3 58.598.5 56.0 98.6 45.3 99.2 32.9 99.4 0.6 67.1 97.6 62.3 98.0 59.2 98.356.5 98.5 45.7 98.8 33.3 99.2 0.7 67.1 97.4 63.3 97.9 61.5 98.1 57.898.3 46.0 98.6 33.9 98.8 0.8 67.4 97.4 64.0 97.7 62.4 97.9 60.1 98.046.7 98.5 35.6 98.6 0.9 67.5 97.3 65.4 97.6 63.0 97.8 61.2 97.8 47.298.3 35.7 98.6 1.0 67.6 97.1 65.9 97.5 63.6 97.6 61.5 97.7 47.8 98.136.4 98.4 *(A), is Yield of the product, %; **(B), is purity of theproduct (Reb A content), %.Purification of Rebaudioside D after Isolation of Rebaudioside a by aOne-Stage Process

When the Rebaudioside A content in the initial extract is not less than40%, and the Rebaudioside D and/or Rebaudioside B combined contents arelower than 1.8%, a one-stage process can be performed in order toprepare highly purified Rebaudioside A. For that purpose Stevia extractwas dissolved in an ethanol-water solution at 50-70° C., preferably55-60° C., for about 10-30 min, preferably 15-20 min, and then at 15-40°C., preferably 20-22° C., for about 18-48 hours, preferably 20-24 hours,with agitation. When the temperature reached 22° C., 1-3 vol. % ofhighly purified Rebaudioside A was added to the reaction mixture as astarter to initiate crystallization. The proportion of extract andethanol depended on content of minor compounds, especially RebaudiosideB and Rebaudioside D, and was between 1.0:2.0-1.0:4.0, w/v, preferably1.0:2.5-3.5, w/v. Ethanol concentration was between 80-90%, preferably82-88%. At lower concentrations of ethanol, a lower ratio of ethanol tosolid can be used, and vice versa. At 89-90% ethanol concentrations, thepurity of Rebaudioside A was more than 97.0% with yields between 13-35%(TABLE 60).

TABLE 60 Yield (Y) and purity of product (RebA), %%, at different ratioof ethanol to extract (v/w) RebA, RebA, RebA, RebA, RebA, RebA, RebA,EtOH, Y, % % Y, % % Y, % % Y, % % Y, % % Y, % % Y, % % % 1:1.75 1:2.01:2.5 1:3.0 1:3.5 1:3.65 1:4.0 80.0 25.2 98.5 20.4 98.7 20.0 98.7 18.299.1 17.8 99.4 17.1 99.5 13.2 99.6 81.0 25.8 98.2 24.2 98.5 22.6 98.721.1 98.9 20.4 99.2 18.3 99.4 13.6 99.4 82.0 27.0 97.7 26.3 98.4 25.798.5 24.5 98.7 23.3 99.1 22.2 99.0 14.1 99.4 83.0 28.3 97.4 27.2 98.426.8 98.1 26.0 98.5 25.2 98.8 24.6 99.0 17.9 99.1 84.0 30.5 97.1 29.697.1 29.5 97.9 28.8 98.4 28.1 98.6 27.7 98.7 20.8 98.7 85.0 35.1 96.733.8 96.6 31.1 97.7 30.4 98.2 29.5 98.5 29.1 98.6 25.6 98.7 86.0 36.296.5 33.9 96.2 32.5 97.5 31.7 97.7 30.6 98.3 30.0 98.6 28.8 98.6 87.040.4 96.1 34.2 95.7 34.2 96.3 33.5 97.5 32.7 97.9 31.6 98.2 29.4 98.488.0 42.5 94.7 35.4 94.5 34.8 94.8 34.1 97.3 33.3 97.8 32.5 97.9 30.698.1 89.0 42.9 92.9 39.7 93.8 38.4 94.2 37.8 94.7 37.1 96.6 37.6 97.435.3 97.6 90.0 43.4 92.1 41.2 93.5 40.7 93.9 40.1 94.5 39.4 96.5 38.596.8 38.8 97.1 RebA content in the initial extract was 48.5%; RebD -0.4%

The precipitate was separated by filtration or centrifugation, washedwith about two volumes of absolute ethanol and dried. Any type ofequipment which allows the separation of precipitate from liquid, suchas various centrifuges or filtration systems, can be used in this stage.Different type of dryers, such as a rotary vacuum dryer, fluid beddryer, rotary tunnel dryer or plate dryer, are suitable to produceRebaudioside A in powder form.

If the initial extract contains a high amount of Rebaudioside B andRebaudioside D for the purification of Rebaudioside A, lowerconcentrations of ethanol and a higher ratio of ethanol to the extractis used (TABLE 61; TABLE 62). Rebaudioside A content in the initialextract was 48.7%.

TABLE 61 Ratio ethanol Purity of product at different RebB content,Ethanol, to solid, % (RebD content was 0.4%) % v/w 0% 0.4% 0.8% 1.1%81.0 2.5 98.7 98.5 98.2 97.9 3.0 98.9 98.7 98.4 98.1 3.5 99.2 98.9 98.698.4 83.0 2.5 98.1 98.2 98.0 97.7 3.0 98.5 98.4 98.2 97.9 3.5 98.8 98.698.4 98.2 85.0 2.5 97.7 97.6 97.4 97.2 3.0 98.2 97.9 97.6 97.4 3.5 98.598.2 97.8 97.6 87.0 2.5 96.3 96.2 97.2 96.8 3.0 97.5 97.6 97.4 97.0 3.597.9 97.9 97.6 97.2

The yield of purified Rebaudioside A can be increased using ethanol forafter-precipitation. For that purpose the temperature of the extractsolution was adjusted to 35-45° C., preferably 37-40° C., and maintainedfor about 10-20 hours, preferably 12-15 hours, and then at 10-25° C.,preferably 20-23° C., for about 10-20 hours, preferably 12-15 hours,with continuous agitation. Then, 0.5-1.0, v/w, preferably 0.5-0.8, v/w,of absolute ethanol to the initial solids, was added to the mixture andthe process was continued for another 12-14 hours. The yields and purityof the product from an extract with a 49.3% Rebaudioside A content aresummarized in TABLE 63.

TABLE 62 Ratio ethanol Purity of product at different content of RebD,Ethanol, to solid, % (RebB content was 0.1%) % v/w 0.5% 1.2% 1.7% 2.6%81.0 2.5 98.7 98.0 97.5 97.1 3.0 98.9 98.3 98.0 97.4 3.5 99.2 98.5 98.297.7 83.0 2.5 98.1 97.7 97.3 97.0 3.0 98.5 98.1 97.8 97.4 3.5 98.8 98.498.0 97.6 85.0 2.5 97.7 97.5 97.1 96.8 3.0 98.2 97.8 97.6 97.1 3.5 98.598.1 97.8 97.2 87.0 2.5 96.3 96.2 95.8 94.2 3.0 97.5 97.3 96.7 96.1 3.597.9 97.6 97.4 96.9

TABLE 63 Additional Yield and purity of RebA at different concentrationsof ethanol ethanol (Ratio of ethanol to extract = 1:3.5, w/v) volume,85% 86% 87% 88% v/w to Yield, RebA, Yield, RebA, Yield, RebA, Yield,RebA, solids % % % % % % % % 0 29.5 98.5 30.6 98.3 32.7 97.9 33.3 97.80.5 31.4 98.5 31.6 98.2 33.4 97.9 33.8 97.6 0.6 32.3 98.2 32.7 98.2 34.397.8 34.7 97.6 0.7 33.5 97.9 33.9 97.7 35.4 97.6 35.9 97.5 0.8 34.1 97.935.2 97.7 36.3 97.6 36.7 97.4 0.9 34.3 97.8 35.4 97.6 36.7 97.5 37.497.4 1.0 34.5 97.8 35.7 97.5 36.9 97.4 37.7 97.2

When the process was carried out without a cooling stage, the purity ofRebaudioside A was higher; however, it resulted in a lower yield of theproduct. The quality of the product increased at higher washingtemperatures. The results obtained using 3.5 volumes of 85% ethanol toone part of extract after 24 hours, with and withoutafter-precipitation, are summarized in TABLE 64.

TABLE 64 Yield, % Content of RebA With after- With after- Withoutprecipitation Without precipitation Temperature, after- (0.8 vol. after-(0.8 vol. ° C. precipitation EtOH) precipitation EtOH) 22.0 29.6 33.598.2 98.5 30.0 28.7 32.8 98.4 98.6 35.0 27.5 32.2 98.7 98.9 40.0 27.031.4 98.8 99.2 45.0 25.4 28.9 99.0 99.4 50.0 24.3 25.6 99.2 99.5 RebA,RebB and RebD contents were 51.3, 0.2% and 0.7%, respectively.

When the content of Rebaudioside A in the final product was less than97% the product was subjected to additional washing as described above.

The filtrate and wash liquid were combined and spray dried. Purificationof Rebaudioside D and a high purity mixture of steviol glycosides wasdeveloped using the same methodology used for other types of remainingsolutions.

The typical compositions of the final product and mother liquor in thisstage are summarized in TABLE 65.

TABLE 65 Steviol glycosides, % Product St RebA RebC RebD RebB RebE RebFStBio DulA Final <0.5 >97.0 <0.5 <0.8 <0.2 <0.3 <0.5 <0.1 <0.2 productMother 45-57.0 15-25.0 15-23.0 0.2-3.7 0-0.5 0.4-1.5 1.5-5.0 0-0.50.2-4.0 liquor

The one-stage purification scheme of Rebaudioside A and the isolation ofa high Rebaudioside D fraction from the remaining solution is presentedin FIG. 7.

Thus, when the initial Stevia extract is distinguished with a highcontent of Rebaudioside A, the initial stage of the reprocessing maystart with the purification of this main compound. The suitabletechnological scheme can be chosen in accordance with the content ofother compounds, especially Rebaudioside B and Rebaudioside D.

The production of highly purified Rebaudioside D was carried out eitherfrom crystals of Rebaudioside A or remaining solutions.

Properties of Rebaudioside D

The high purity Reb D obtained in this invention, having a molecularweight of 1129.15, a molecular formula of C₅₀H₈₀O₂₈, and the structurepresented in FIG. 3, is in the form of a white and odorless powder. Thecompound is about 180-200 times sweeter than sugar when compared to a10% sucrose solution. The infrared absorption spectrum is shown in FIG.16. Reb D exhibits a characteristic absorption maximum at around 1730cm⁻¹.

Other properties of the pure Reb D compound include a melting point of248-249° C., and a specific rotation of [α]^(D)−29.5° in 50% ethanol(C=1.0). The solubility of Reb D in water is around 0.2%, and increaseswith an increase in temperature. Reb D precipitates upon cooling of thesolution. It is highly soluble during the chromatographic separationstage and before crystallizing.

Reb D is soluble in diluted solutions of methanol, ethanol, n-propanol,and isopropanol. However, it is insoluble in acetone, benzene,chloroform, and ether.

Reb D obtained in accordance with the present invention is heat andpH-stable.

Reb D obtained according to this invention may be incorporated as a highintensity natural sweetener in foodstuffs, beverages, pharmaceuticalcompositions, cosmetics, chewing gums, table top products, cereals,dairy products, toothpastes and other oral cavity compositions, etc. Theexamples which follow show representative proportions which may beemployed.

Reb D as a sweetening compound may be employed as the sole sweetener, orit may be used together with other naturally occurring high intensitysweeteners such as Stevioside, Reb A, Reb B, Reb C, Reb E, Reb F,Steviolbioside, Dulcoside A, Rubusoside, mogrosides, brazzein,neohesperidin dihydrochalcone, glycyrrhizic acid and its salts,thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside,phlomisoside−1, dimethyl-hexahydrofluorene-dicarboxylic acid,abrusosides, periandrin, carnosiflosides, cyclocarioside,pterocaryosides, polypodoside A, brazilin, hernandulcin, phillodulcin,glycyphyllin, phlorizin, trilobatin, dihydroflavonol,dihydroquercctin-3-acetate, neoastilibin, trans-cinnamaldehyde, monatinand its salts, selligueain A, hematoxylin, monellin, osladin,pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin,curculin, neoculin, chlorogenic acid, cynarin, Luo Han Guo sweetener,siamenoside and others.

Reb D may also be used in combination with synthetic high intensitysweeteners such as sucralose, potassium acesulfame, aspartame, alitame,saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dulcin,suosan,N—[N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester,N—[N-[3-(3-hydroxy-4-methoxyphenyl)-3-methylbutyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester,N—[N-[3-(3-methoxy-4-hydroxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine1-methyl ester, salts thereof, and the like.

Moreover, Reb D can be used in combination with natural sweetenersuppressors such as gymnemic acid, hodulcin, ziziphin, lactisole, andothers. Reb D may also be combined with various umami taste enhancers.Reb D can be mixed with umami tasting and sweet aminoacids such asglutamate, aspartic acid, glycine, alanine, threonine, proline, serine,glutamate, and tryptophan.

Reb D may also be combined with polyols or sugar alcohols. The term“polyol” refers to a molecule that contains more than one hydroxylgroup. A polyol may be a diol, triol, or a tetraol which contain 2, 3,and 4 hydroxyl groups, respectively. A polyol also may contain more thanfour hydroxyl groups, such as a pentaol, hexaol, heptaol, or the like,which contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, apolyol also may be a sugar alcohol, polyhydric alcohol, or polyalcoholwhich is a reduced form of carbohydrate, wherein the carbonyl group(aldehyde or ketone, reducing sugar) has been reduced to a primary orsecondary hydroxyl group. Examples of polyols include, but are notlimited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol,inositol, isomalt, propylene glycol, glycerol, threitol, galactitol,hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reducedxylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltosesyrup, reduced glucose syrup, hydrogenated starch hydrolyzates,polyglycitols and sugar alcohols or any other carbohydrates capable ofbeing reduced which do not adversely affect the taste of the sweetenercomposition.

Reb D may be combined with reduced calorie sweeteners such asD-tagatose, L-sugars, L-sorbose, L-arabinose, and others.

Reb D may also be combined with various carbohydrates. The term“carbohydrate” generally refers to aldehyde or ketone compoundssubstituted with multiple hydroxyl groups, of the general formula(CH₂O)_(n), wherein n is 3-30, as well as their oligomers and polymers.The carbohydrates of the present invention can, in addition, besubstituted or deoxygenated at one or more positions. Carbohydrates, asused herein, encompass unmodified carbohydrates, carbohydratederivatives, substituted carbohydrates, and modified carbohydrates. Asused herein, the phrases “carbohydrate derivatives”, “substitutedcarbohydrate”, and “modified carbohydrates” are synonymous. Modifiedcarbohydrate means any carbohydrate wherein at least one atom has beenadded, removed, or substituted, or combinations thereof. Thus,carbohydrate derivatives or substituted carbohydrates includesubstituted and unsubstituted monosaccharides, disaccharides,oligosaccharides, and polysaccharides. The carbohydrate derivatives orsubstituted carbohydrates optionally can be deoxygenated at anycorresponding C-position, and/or substituted with one or more moietiessuch as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino,amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino,alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl,sulfenyl, sullinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl,phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino,hydrazino, carbamyl, phospho, phosphonato, or any other viablefunctional group provided the carbohydrate derivative or substitutedcarbohydrate functions to improve the sweet taste of the sweetenercomposition.

Examples of carbohydrates which may be used in accordance with thisinvention include, but are not limited to, tagatose, trehalose,galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides,various types of maltodextrins, dextran, sucrose, glucose, ribulose,fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose,idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose,isomaltulose, erythrose, deoxyribose, gulose, idose, talose,erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin,glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid,glucono-lactone, abequose, galactosamine, beet oligosaccharides,isomalto-oligosaccharides (isomaltose, isomaltotriose, panose and thelike), xylo-oligosaccharides (xylotriose, xylobiose and the like),xylo-terminated oligosaccharides, gentio-oligosaccharides (gentiobiose,gentiotriose, gentiotetraose and the like), sorbose,nigero-oligosaccharides, palatinose oligosaccharides,fructooligosaccharides (kestose, nystose and the like), maltotetraol,maltotriol, malto-oligosaccharides (maltotriose, maltotetraose,maltopentaose, maltohexaose, maltoheptaose and the like), starch,inulin, inulo-oligosaccharides, lactulose, melibiose, raffinose, ribose,isomerized liquid sugars such as high fructose corn syrups, couplingsugars, and soybean oligosaccharides. Additionally, the carbohydrates asused herein may be in either the D- or L-configuration.

Reb D obtained according to this invention can be used in combinationwith various physiologically active substances or functionalingredients. Functional ingredients generally are classified intocategories such as carotenoids, dietary fiber, fatty acids, saponins,antioxidants, nutraceuticals, flavonoids, isothiocyanates, phenols,plant sterols and stanols (phytosterols and phytostanols); polyols;prebiotics, probiotics; phytoestrogens; soy protein; sulfides/thiols;amino acids; proteins; vitamins; and minerals. Functional ingredientsalso may be classified based on their health benefits, such ascardiovascular, cholesterol-reducing, and anti-inflammatory.

Reb D obtained according to this invention may be applied as a highintensity sweetener to produce zero calorie, reduced calorie or diabeticbeverages and food products with improved taste characteristics. It mayalso be used in drinks, foodstuffs, pharmaceuticals, and other productsin which sugar cannot be used. In addition, Reb D can be used as asweetener not only for drinks, foodstuffs, and other products dedicatedfor human consumption, but also in animal feed and fodder with improvedcharacteristics.

Examples of products in which Reb D may be used as a sweetening compoundinclude, but are not limited to, alcoholic beverages such as vodka,wine, beer, liquor, and sake, etc.; natural juices; refreshing drinks;carbonated soft drinks; diet drinks; zero calorie drinks; reducedcalorie drinks and foods; yogurt drinks; instant juices; instant coffee;powdered types of instant beverages; canned products; syrups; fermentedsoybean paste; soy sauce; vinegar; dressings; mayonnaise; ketchups;curry; soup; instant bouillon; powdered soy sauce; powdered vinegar;types of biscuits; rice biscuit; crackers; bread; chocolates; caramel;candy; chewing gum; jelly; pudding; preserved fruits and vegetables;fresh cream; jam; marmalade; flower paste; powdered milk; ice cream;sorbet; vegetables and fruits packed in bottles; canned and boiledbeans; meat and foods boiled in sweetened sauce; agricultural vegetablefood products; seafood; ham; sausage; fish ham; fish sausage; fishpaste; deep fried fish products; dried seafood products; frozen foodproducts; preserved seaweed; preserved meat; tobacco; medicinalproducts; and many others. In principle it can have unlimitedapplications.

During the manufacturing of products such as foodstuffs, drinks,pharmaceuticals, cosmetics, table top products, and chewing gum, theconventional methods such as mixing, kneading, dissolution, pickling,permeation, percolation, sprinkling, atomizing, infusing and othermethods may be used.

Moreover, the sweetener obtained in this invention may be used in dry orliquid forms. It can be added before or after heat treatment of foodproducts. The amount of the sweetener depends on the purpose of usage.As discussed above, it can be added alone or in combination with othercompounds.

EXAMPLES

The following examples illustrate preferred embodiments of the inventionfor the isolation and purification of Reb D and related compounds andthe use thereof in foodstuffs and pharmaceuticals. It will be understoodthat the invention is not limited to the materials, proportions,conditions and procedures set forth in the examples, which are onlyillustrative.

Example 1: Purification of Reb D from Stevia rebaudiana Bertoni PlantLeaves

Two kg of Stevia rebaudiana Bertoni plant leaves were dried at 45° C. toan 8.0% moisture content and ground to 10-20 mm particles. The contentof different glycosides in the leaves was as follows: Stevioside—2.55%,Reb A—7.78%, Reb B—0.01%, Reb C—1.04%, Reb D—0.21%, Reb E—0.02%, RebF—0.14%, Dulcoside A—0.05%, and Steviolbioside—0.05%. The total contentof steviol glycosides was 11.85%. The dried material was loaded into acontinuous extractor and the extraction was carried out with 40.0 L ofwater at a pH of 6.5 at 40° C. for 160 min. The filtrate was collectedand subjected to chemical treatment. Calcium oxide in the amount of 400g was added to the filtrate to adjust the pH within the range of8.5-9.0, and the mixture was maintained for 15 min with slow agitation.Then, the pH was adjusted to around 3.0 by adding 600 g of FeCl₃ and themixture was maintained for 15 min with slow agitation. A small amount ofcalcium oxide was further added to adjust the pH to 8.5-9.0 and themixture was maintained for 30 min with slow agitation. The precipitatewas removed by filtration on a plate-and-frame filter press using cottoncloth as the filtration material. The slightly yellow filtrate waspassed through the column, packed with cation-exchange resin AmberliteFCP22 (H⁺) and then, through the column with anion-exchange resinAmberlite FPA53 (OH⁻). The flow rate in both columns was maintained atSV=0.8 hour⁻¹. After completion both columns were washed with RO waterto recover the steviol glycosides left in the columns and the filtrateswere combined. The portion of combined solution containing 120 g totalsteviol glycosides was passed through seven columns, wherein each columnwas packed with specific macroporous polymeric adsorbent YWD-03(Cangzhou Yuanwei, China). The first column with the size of ⅓ of theothers acted as a “catcher column”. The SV was around 1.0 hour⁻¹. Afterall extract was passed through the columns, the resin sequentially waswashed with 1 volume of water, 2 volumes of 0.5% NaOH, 1 volume ofwater, 2 volumes of 0.5% HCl, and finally with water until the pH was7.0. The “catcher column” was washed separately. Desorption of theadsorbed steviol glycosides was carried out with 52% ethanol at SV=1.0hour⁻¹. Desorption of the first “catcher column” was carried outseparately and the filtrate was not mixed with the main solutionobtained from other columns. Desorption of the last column also wascarried out separately. Eluates from the first to fifth columns werecombined and treated separately.

The combined solution of steviol glycosides was mixed with 0.3% ofactivated carbon from the total volume of solution. The suspension wasmaintained at 25° C. for 30 min with continuous agitation. Separation ofcarbon was carried out on a press-filtration system. For additionaldecolorization the filtrate was passed through the columns packed withcation-exchange resin Amberlite FCP22 (H⁺) followed with anion-exchangeresin Amberlite FPA53 A30B (OH⁺). The flow rate in both columns wasaround SV=0.5 hour⁻¹. The ethanol was distilled using a vacuumevaporator. The solids content in the final solution was around 15%. Theconcentrate was passed through the columns packed with cation-exchangeresin Amberlite FCP22 (H⁺) and anion-exchange resin Amberlite FPA53(OH⁻) with SV=0.5 hour⁻¹. After all the solution was passed through thecolumns, both resins were washed with RO water to recover the steviolglycosides left in the columns. The resulting refined extract wastransferred to the nano-filtration device, concentrated to around 52% ofsolids content and spray dried to obtain a highly purified mixture ofsteviol glycosides. The yield was 100.7 g with 96.8% content of TSG. Themixture contained Stevioside—21.5%, Reb A—65.5%, Reb B—0.1%, Reb C—9.0%,Reb D—0.4%, Reb E—0.5%, Reb F—2.4%, Dulcoside A—0.5%, andSteviolbioside—0.1%.

The combined eluate from the last column, containing 13.3 g of steviolglycosides and around 28.4% Reb D, was deionized and decolorized asdiscussed above and concentrated to a 33.5% content of total solids.

The concentrate was mixed with two volumes of anhydrous methanol andmaintained at 20-22° C. for 24 hours with intensive agitation.

The resulting precipitate was separated by filtration and washed withabout two volumes of absolute methanol. The yield of Reb D was 3.9 gwith around 86% purity.

For the further purification the precipitate was suspended in threevolumes of 60% methanol and treated at 55° C. for 30 min, then cooleddown to 20-22° C. and agitated for another 2 hours.

The resulting precipitate was separated by filtration and washed withabout two volumes of absolute methanol and subjected to similartreatment with a mixture of methanol and water.

The yield of Reb D was 3.37 g with 99.3% purity.

The purity of the Rebaudioside D was determined using HPLC which wasperformed using a ZORBAX NH₂ column (150×4.6 mm, 5 μm) at a temperatureof 30° C. The mobile phase comprised a solution of 20% buffer (0.0125%acetic acid and 0.0125% ammonium acetate) and 80% acetonitrile at a flowrate of 1.0 mL/min. 12 μL of each sample was injected in duplicate andthe sample was analyzed using a UV detector at 210 nm (4 nm bandwidth)with a reference of 260 nm (100 nm bandwidth). The analysis required arun time ranging from 40 to 60 min.

A buffer solution of 0.0125% acetic acid and 0.0125% ammonium acetatewas prepared by dissolving 0.125 g ammonium acetate and 125 μL glacialacetic acid in one liter of water. The retention time of Rebaudioside Bwas adjusted by varying the ratio of ammonium acetate to acetic acidwhile maintaining a total of 0.025% of both combined. Increasing theamount of acetic acid decreased the retention time of Rebaudioside B.

A diluent solution was prepared by mixing 500 mL of ethyl alcohol and500 mL of the buffer solution. Rebaudioside D standards were prepared bydiluting 10.0±0.5 mg (recorded to the nearest 0.1 mg) of theRebaudioside D standard with 4 mL of the diluent solution to make astandard solution of approximately 2500 mg/L. The Rebaudioside Dstandard solution was injected at 10.8, 11.4, 12.6 and 13.2 μL. Themoisture content was measured by Karl Fischer analysis every time astandard was prepared and corrections were made based on the solventpurity according to the certificate of analysis.

Stevioside standards were prepared by diluting 12.5±0.5 mg (recorded tothe nearest 0.1 mg) of the stevioside standard with 5 mL of the diluentsolution to make a standard solution of approximately 2500 mg/L standard(stock A) (correcting for moisture and purity). The stevioside standardwas then diluted using one mL of stock A to ten mL of diluent to producea 250 mg/L standard (stock B), and stock standards were diluted to finalconcentrations ranging from 2.5 to 50 mg/L.

Samples of the Rebaudioside D compositions were prepared by diluting125±2 mg (recorded to the nearest 0.1 mg) of the Rebaudioside Dcomposition with 50 mL of the diluent solution to make a sample solutionof approximately 2500 mg/L (correcting for moisture). Individuallyprepared duplicate samples were injected at 12 μL. If the samples werenot analyzed immediately, they were stored without headspace, undernitrogen, and desiccated.

The TABLE 66 provides a guideline for retention times for Rebaudioside Dand other steviol glycosides. However, those of ordinary skill in theart should appreciate that the retention times may be modified asneeded.

TABLE 66 Compound HPLC retention time, min Stevioside 5.4 Rebaudioside A7.8 Rebaudioside B 28.6 Rebaudioside C 6.0 Rebaudioside D 15.7Rebaudioside E 10.7 Rebaudioside F 6.4 Steviolbioside 17.7 Dulcoside A4.5 Rubusoside 3.0

Example 2: Purification of Reb D and Reb A from Stevia Extract

One kg of Stevia extract containing Stevioside—22.63%, Reb A—52.46%, RebC—7.52%, Reb D—1.18%, Reb B—0.55%, Reb E—0.64%, Reb F—1.79%,Steviolbioside—0.1%, and Dulcoside A—1.59%, with 88.46% of total steviolglycosides content, was dissolved in 3000 mL of 95% ethyl alcohol andmaintained at 80° C. for 35 min, and then at 15° C. for 12 hours withagitation. When the temperature reached 22° C., 1.0% of highly purifiedReb A was added to the reaction mixture as a starter to initiatecrystallization.

Precipitate was separated by filtration and washed with about twovolumes of 99.5% ethanol.

The yield of crystalline material was 47.1% with content of 81.7% and3.3% of Reb A and Reb D respectively.

The precipitate was mixed with 5 volumes of 78% ethanol and incubated at50° C. for 3 hours with agitation. Then, the mixture was cooled down toroom temperature and the precipitate with 21.4% Reb D content wasseparated by filtration.

The remaining solution after isolation of Reb D was mixed with 1% Reb Aas starter and left for crystallization at 22° C. for 12 hours. Thecrystals were separated by filtration and washed with about two volumesof ethanol. Reb A content in the crystals was 98.8%.

For further purification the high RebD precipitate was suspended in 50%ethanol at 1:2 wt/vol. ratio, and maintained for 12 hours at 35° C. withagitation. The suspension was filtered and precipitate was dried. Theyield of precipitate with 21.7% of Reb A and 81.6% of Reb D contents was35.5% from initial material. The precipitate was subjected to similartreatment to get a product with 3.7% of Reb A and 95.7% of Reb Dcontent. The yield of this product was 58.5%.

The obtained Reb D was dissolved in 2 volumes of 30% methanol andtreated with 0.3% of activated carbon at 60° C. for 30 min thensubjected to hot filtration. Reb D spontaneously precipitated afterfiltration.

Highly purified Reb D (98.4%) was separated by filtration and dried at80° C. for 12 hours.

Example 3: Purification of Reb D and Reb A from Stevia Extract

One kg of Stevia extract containing Stevioside—22.63%, Reb A—52.46%, RebC—7.52%, Reb D—1.18%, Reb B—0.55%, Reb E—0.64%, Reb F—1.79%,Steviolbioside—0.1%, and Dulcoside A—1.59%, with 88.46% of total steviolglycosides content, was dissolved in 3000 mL of 88% ethyl alcohol andmaintained at 55° C. for 15 min, and then at 22° C. for 24 hours withagitation. When the temperature reached 22° C., 1.0% of highly purifiedReb A was added to the mixture as a starter to initiate crystallization.The precipitate was separated by filtration and washed with about twovolumes of 99.5% ethanol.

The yield of crystalline material was 33.0% with content of 95.6% and3.5% of Reb A and Reb D respectively.

The precipitate was mixed with 3.0 volumes of anhydrous methanol andincubated at 22° C. for 90 minutes, and the precipitate with content of98.9% Reb A was separated by filtration, washed with about two volumesof anhydrous methanol and dried at 80° C. for 12 hours. The yield of theproduct from the initial extract was 29.3%.

Methanol was removed by vacuum evaporation and the liquid wasconcentrated to 33.5% solids.

The obtained syrup with 28.3% Reb D content was mixed with two volumes(vol/vol.) of methanol, and 1% of pure Reb D was added to start thecrystallization process. The mixture was maintained at 22° C. for 24hours with agitation.

The precipitate was separated by filtration and washed with about twovolumes of anhydrous methanol and dried. The yield of the product was10.4 g with 84.5% content of Reb D.

To achieve further purification, the precipitate was suspended in 60%methanol at 1:3, wt/vol. ratio, and maintained at 55° C. for 15 minutes,then cooled down to 22° C. and agitated for another 1.5 hours. Thesuspension was filtered. The precipitate was washed with about twovolumes of anhydrous methanol and dried at 80° C. for about 12 hours.

The yield of precipitate with 99.1% of Reb D content was 9.0 g.

Example 4: Purification of Reb D from Remaining Solution after Isolationof Reb A

One kg of Stevia extract containing Stevioside—24.3%, Reb A—62.1%, RebC—8.8%, Reb D—1.6%, Reb B—0.2%, Reb E—0.6%, Reb F—1.0%,Steviolbioside—0.10%, and Dulcoside A—1.3%, with 87.9% of total steviolglycosides content, was dissolved in 3000 mL of 85% methyl alcohol andmaintained at 55° C. for 15 min, and then at 22° C. for 1.5 hours withagitation. When the temperature reached 22° C., 1.0% of highly purifiedReb A was added to the reaction mixture as a starter to initiatecrystallization.

The precipitate was separated by filtration and washed with about twovolumes of anhydrous 99.5% methanol.

The yield of crystalline material was 42.5% with content of 98.5% and0.3% of Reb A and Reb D respectively.

The combined total filtrate containing Stevioside (49.6%), Reb C(17.5%), Reb A (29.9%) and Reb D (3.0%) as the main substances wasevaporated in order to remove methanol and concentrated up to a totalsolids content of about 10.0%.

The remaining solution, containing 115 g of total solids after isolationof Reb A, was passed through a series of eight columns with the rateequal to 1.0 bed volume (BV) per 1 hour. Each column was packed with 150mL of specific polar macroporous polymeric resin. Upon completion of theadsorption the resin was washed with 5 volumes of water and desorptionof the adsorbed steviol glycosides was carried out with 50-52% ethanolat BV=1.0-2.0 hour⁻¹.

Eluates from the last two columns, containing around 80% of totalquantity of Reb D, were collected separately. Ethanol was removed byevaporation under vacuum and dried.

The operation was repeated several times to complete treatment of allremaining solution.

The average concentration and total quantity of Reb D was 21.4% and 10.1g respectively.

For the sake of further purification, the material was suspended in 60%methanol at 1:3 wt/vol. ratio, and maintained at 55° C. for 15 minutes,cooled down to 22° C. and agitated for another 1.5 hours. The suspensionwas filtered; the precipitate was washed with about two volumes ofanhydrous methanol.

The operation was repeated two times and the final product was dried at80° C. for about 12 hours.

The yield of precipitate with 98.6% of Reb D contents was 9.4 g.

The remaining solution after isolation of Reb A and Reb D contained amixture of steviol glycosides with a TSG content of 96.7%.

Example 5: Purification of Reb D from Remaining Solution after Isolationof Reb A

One kg of Stevia extract containing Stevioside (32.3%), Reb A (52.6%),Reb C (9.6%), Reb D (2.3%), Reb B (0.1%), Reb E (0.6%), Reb F (1.6%),Steviolbioside (0.1%), and Dulcoside A (0.8%) on a dry basis, with 86.3%of total steviol glycosides content, was dissolved in 3000 mL of 99.5%ethyl alcohol and maintained at 80° C. for about 10 min. During theheating crude Reb A was precipitated. The suspension was cooled down to22° C. and maintained for 10 hours with agitation. The precipitate wasseparated by filtration and washed with 600 mL of 99.5% ethanol.

The obtained white precipitate contained Stevioside (7.55%), Reb A(79.87%), Reb C (7.76%), Reb D (1.55%), Reb B (0.14%), Reb E (0.41%),Reb F (2.25%), Steviolbioside (0.12%), and Dulcoside A (0.35%) on a drybasis. The total steviol glycosides content was 99.5%. The yield of theproduct was 56.4% from the initial extract.

The filtrate and washed liquid were combined and spray dried. Theproduct contained Stevioside (64.32%), Reb A (17.32%), Reb C (11.98%),Reb D (3.27%), Reb B (0.05%), Reb E (0.85%), Reb F (0.76%),Steviolbioside (0.07%), and Dulcoside A (1.38%) on a dry basis. Around436 g was obtained and it was named as RS-1.

The wet cake of crude Reb A was suspended in 2.3 volumes (wt/vol.) of80.5% ethanol and completely dissolved by heating at 80° C. for about 30min with continuous agitation. The solution was cooled to 20° C. andpure Reb A was added in the amount of 1.0% from solids. The cooling wascontinued to 15° C. and maintained for 12 hours with slow agitation.Then 1.0 volume (vol/wt) of 99.5% ethanol to the initial solids wasadded to the crystallization mixture and the suspension was maintainedat the same temperature for another 12 hours.

The precipitate was separated by filtration and washed with about 850 mLof 99.5% ethanol. The obtained white precipitate contained Stevioside(1.1%), Reb A (95.3%), Reb C (1.3%), Reb D (1.28%), Reb B (0.10%), Reb E(0.32%), Reb F (0.4%), Steviolbioside (0.10%), and Dulcoside A (0.1%) ona dry basis. The yield of the product was 66.4% (374 g) from the initialsolids at the first stage.

The filtrate and washing liquid were combined and spray dried. Theproduct contained Stevioside (20.25%), Reb A (49.49%), Reb C (20.48%),Reb D (2.08%), Reb B (0.22%), Reb E (0.59%), Reb F (5.89%),Steviolbioside (0.16%), and Dulcoside A (0.84%) on a dry basis. Theyield was 190 g and it was named as RS-2.

The further purification of the obtained product was carried outsimilarly to the second step described above with the application of 3.5volumes (vol/wt) of 77.7% ethanol as the crystallization medium.

The crystals were separated and dried in a vacuum oven at 80° C. for12-14 hours under reduced pressure. The product contained Stevioside(<0.1%), Reb A (98.71%), Reb C (<0.1%), Reb D (0.72%), Reb B (<0.10%),Reb E (0.12%), and Reb F (0.15%) on a dry basis. The yield of theproduct was 75.4% from the initial solids at the first stage. 282.0 g ofproduct was obtained.

The spray dried mother liquor contained Stevioside (4.17%), Reb A(84.85%), Reb C (4.98%), Reb D (3.00%), Reb B (0.10%), Reb E (0.93%),Reb F (1.17%), Steviolbioside (0.41%), and Dulcoside A (0.41%) on a drybasis. The yield of the product was 92 g and it was named as RS-3.

Isolation and purification of Reb D from RS-1 was carried out similarlyto the procedure described in EXAMPLE 4 for remaining solution. 145 g ofthe material was dissolved in 580 mL of water and the resulted 20%solution was passed through the series of ten columns with the rateequal to 1.0 bed volume (BV) per 1 hour. Each column was packed with 150mL of specific polar macroporous polymeric resin. Upon completion of theadsorption the resin was washed with 5 volume of water and desorption ofthe adsorbed steviol glycosides was carried out with 50-52% ethanol atBV=1.0-2.0 hour⁻¹.

Eluates from the last two columns, containing around 80% of the totalquantity of Reb D, were collected separately. The combined solution wasmixed with 0.3% activated carbon from the total volume of solution. Thesuspension was maintained at 25° C. for 30 min with continuous agitationand carbon was separated by filtration.

The filtrate was evaporated and dried under vacuum.

The operation was repeated three times to complete the treatment of allRS-1.

The average concentration and total quantity of Reb D was 31.6% and 11.5g respectively.

The resulting material was suspended in 60% methanol at 1:3 wt/vol.ratio, and maintained at 55° C. for 15 minutes, then cooled down to 22°C. and agitated for another 1.5 hours. The suspension was filtered andthe precipitate was washed with about two volumes of anhydrous methanol.

The operation was repeated two times and the final product was dried at80° C. for about 12 hours.

The yield of precipitate with 98.8% of Reb D contents was 9.2 g.

For the purpose of further retreatment, RS-2 was recycled to the firststage of enrichment with Reb A.

Isolation and purification of Reb D from RS-3 was similar to theprocedure described in EXAMPLE 3.

The yield of Reb D was 1.8 g with 99.1% purity.

The yield of Reb A was 65.7 g with 98.5% purity.

Example 6: Purification of Reb D from Remaining Solution after Isolationof Reb A

A wet cake of crude Rebaudioside A and RS-1 was prepared in a mannersimilar to that described in EXAMPLE 5.

The wet cake of crude Reb A was suspended in 2.65 volumes (wt/vol.) of75.0% ethanol and completely dissolved by heating at 80° C. for about 30min with continuous agitation. The solution was cooled to 20° C. andpure Reb A was added in the amount of 1.0% from solids. The cooling wascontinued to 15° C. and maintained for 12 hours with slow agitation.Then 0.8 volume (vol/wt) of 99.5% ethanol to the initial solids wasadded to the crystallization mixture and the suspension was maintainedat the same temperature for another 12 hours.

The precipitate was separated by filtration and washed with about 550 mLof 99.5% ethanol. The product contained Stevioside (0.41%), Reb A(98.05%), Reb C (0.22%), Reb D (0.61%), Reb B (0.11%), Reb E (0.24%),Reb F (0.26%), and Dulcoside A (0.10%) on a dry basis. The yield of theproduct was 369 g or 65.4% from the initial solids.

The filtrate was spray dried to make RS-2 which contained Stevioside(21.05%), Reb A (45.51%), Reb C (22.01%), Reb D (3.33%), Reb B (0.20%),Reb E (0.73%), Reb F (6.00%), Steviolbioside (0.35%), and Dulcoside A(0.82%) on a dry basis. The yield of product was 195 g. The product wasrecycled to the first stage of the Reb A isolation process.

The isolation and purification of Reb D from RS-1 was carried out in amethod similar to that described in EXAMPLE 5.

The yield of precipitate with 99.1% of Reb D contents was 8.9 g.

Example 7: Purification of Reb D from Remaining Solution after Isolationof Reb A

One kg of Stevia extract containing Stevioside (37.1%), Reb A (48.2%),Reb C (10.2%), Reb D (0.9%), Reb B (0.1%), Reb E (0.8%), Reb F (1.1%),Steviolbioside (0.1%), and Dulcoside A (1.5%) on a dry basis, with 90.5%of total steviol glycosides content, was dissolved in 3000 mL of 99.5%ethyl alcohol and maintained at 80° C. for about 10 min. During theheating crude Reb A precipitated. The suspension was cooled down to 22°C. and maintained for 10 hours with agitation. The precipitate wasseparated by filtration and washed with 600 mL of 99.5% ethanol.

The obtained white precipitate contained Stevioside (9.06%), Reb A(78.89%), Reb C (9.42%), Reb D (0.61%), Reb B (0.1%), Reb E (0.60%), RebF (0.95%), Steviolbioside (0.1%), and Dulcoside A (0.27%) on a drybasis. Total steviol glycosides content was 98.4%. The yield of theproduct was 53.7% from initial extract.

The filtrate and washing liquid were combined and spray dried (463 g).The product contained Stevioside (69.62%), Reb A (12.60%), Reb C(11.10%), Reb D (1.24%), Reb B (0.10%), Reb E (1.03%), Reb F (1.27%),Steviolbioside (0.10%), and Dulcoside A (2.94%) on a dry basis. 463 g ofproduct (RS-1) was obtained.

The wet cake of crude Reb A was suspended in 3.0 volumes (wt/vol.) of81.0% ethanol. After homogeneous suspension was obtained the temperaturewas increased to 40° C. and maintained for 12 hours. Then, thesuspension was cooled down to 22° C., 0.5 volumes (vol/wt) of 99.5%ethanol was added, and the suspension was maintained for another 12hours with continuous agitation.

The precipitate was separated by filtration and washed with about 600 mLof 99.5% ethanol. The product contained Stevioside (0.18%), Reb A(98.32%), Reb C (0.20%), Reb D (0.51%), Reb B (0.1%), Reb E (0.28%), RebF (0.31%), and Dulcoside A (0.1%) on a dry basis. The yield of theproduct was 54.7% from the initial solids at the first stage. 349.0 g ofhighly purified Reb A was obtained with 34.9% output from initialextract.

The spray dried mother liquor contained Stevioside (25.54%), Reb A(42.82%), Reb C (26.54%), Reb D (0.80%), Reb B (0.10%), Reb E (1.19%),Reb F (2.14%), Steviolbioside (0.29%), and Dulcoside A (0.59%) on a drybasis. 188 g of product was obtained, and was recycled to the firststage of the initial isolation process of Reb A.

Isolation and purification of Reb D from RS-1 was carried out using amethod similar to that described in EXAMPLE 5.

The yield of precipitate with 98.7% of Reb D contents was 3.5 g.

Example 8: Purification of Reb D from Remaining Solution after Isolationof Reb A

One kg of Stevia extract containing Stevioside (22.63%), Reb A (52.46%),Reb C (7.52%), Reb D (1.18%), Reb B (0.55%), Reb E (0.64%), Reb F(1.79%), Steviolbioside (0.1%), and Dulcoside A (1.59%) on a dry basis,with 88.46% of total steviol glycosides content, was dissolved in 3500mL of 85.0% ethyl alcohol at 55° C. for about 15 min. The temperature ofthe solution was adjusted to 40° C. and maintained for 12 hours, andthen at 22° C. for another 12 hours with continuous agitation. When thetemperature reached 22° C., 1 vol. % of highly purified Reb A was addedto the reaction mixture as a starter to initiate crystallization. Then,0.8 volumes (vol/wt) of 99.5% ethanol to the initial solids, was addedto the reaction mixture and the process was continued for another 12hours. The precipitate was separated by filtration and washed with 1000mL of 99.5% ethanol.

The product contained Stevioside (0.31%), Reb A (98.2%), Reb C (0.26%),Reb D (0.3%), Reb B (0.05%), Reb E (0.23%), Reb F (0.55%), and DulcosideA (0.1%) on a dry basis. The yield of the product was 42.0% from theinitial extract.

The spray dried mother liquor contained Stevioside (38.79%), Reb A(19.34%), Reb C (12.78%), Reb D (1.82%), Reb B (0.91%), Reb E (0.94%),Reb F (2.69%), Steviolbioside (0.17%), and Dulcoside A (2.67%) with80.1% of total steviol glycosides content on a dry basis. The yield ofthe product was 580 g.

Isolation and purification of Reb D from RS-1 was carried out using amethod similar to that described in EXAMPLE 5.

The yield of precipitate with 98.9% of Reb D contents was 7.1 g.

Example 9: Purification of Reb D from Remaining Solution after Isolationof Reb A and Steviol Glycosides

One kg of spray dried remaining solution (RS-1) obtained after isolationof Reb A as per the scheme described in EXAMPLE 6, containing Stevioside(64.32%), Reb A (17.32%), Reb C (11.98%), Reb D (3.27%), Reb B (0.05%),Reb E (0.85%), Reb F (0.76%), Steviolbioside (0.07%), and Dulcoside A(1.38%) on a dry basis and with 82.3% total steviol glycosides content,was dissolved in 3000 mL of absolute methyl alcohol and maintained at75° C. for about 10 min. During the heating, crude Steviosideprecipitated. The suspension was cooled down to 25° C. and maintainedfor 10 hours with agitation. The precipitate was separated by filtrationand washed with 900 mL of absolute methanol.

The yield of crystals containing Stevioside (93.2%), Reb A (3.6%), Reb C(2.1%), Reb D (0.2%), Reb B (0.05%), Reb E (0.2%), Reb F (0.2%),Steviolbioside (0.1%), and Dulcoside A (0.35%) was 53.3% from initialmaterial, and the total steviol glycoside content was 97.7% on a drybasis.

The filtrate and washing liquid were combined and spray dried. Thecontent of steviol glycosides was as follows: Stevioside (31.36%), Reb A(32.98%), Reb C (23.26%), Reb D (6.77%), Reb B (0.05%), Reb E (1.59%),Reb F (1.40%), Steviolbioside (0.04%), and Dulcoside A (2.56%). Theyield of the product was around 467 g.

The isolation and purification of Reb D from the spray dried remainingsolution was carried out using a method similar to that described inEXAMPLE 5.

The yield of precipitate with 98.9% of Reb D contents was 20.5 g.

Example 10: Purification of Reb D from Remaining Solution afterIsolation of Reb A and Steviol Glycosides

One kg of Stevia extract containing Stevioside—24.3%, Reb A—62.1%, RebC—8.8%, Reb D—1.6%, Reb B—0.2%, Reb E—0.6%, Reb F—1.0%,Steviolbioside—0.10%, and Dulcoside A—1.3%, with 87.9% of total steviolglycosides content was dissolved in 3000 mL of 85% methyl alcohol andmaintained at 55° C. for 15 min, and then at 22° C. for 1.5 hours withagitation. When the temperature reached 22° C., 1.0% of highly purifiedReb A was added to the reaction mixture as a starter to initiatecrystallization.

The precipitate was separated by filtration and washed with about twovolumes of anhydrous 99.5% methanol.

The yield of crystalline material was 42.5% including Stevioside (0.3%),Reb A (98.5%), Reb C (0.4%), Reb D (0.3%), Reb B (0.1%), Reb E (0.1%),Reb F (0.1%), Steviolbioside (0.1%), and Dulcoside A (0.1%).

The combined total filtrate containing Stevioside (42.0%), Reb A(35.2%), Reb C (15.0%), Reb D (2.6%), Reb B (0.3%), Reb E (1.0%), Reb F(1.7%), Steviolbioside (0.1%), and Dulcoside A (2.2%) was agitated foranother 12 hours at 22° C. after adding 2 g of high purity Steviosidefor the initiation of crystallization.

The yield of crystals containing Stevioside (92.6%), Reb A (3.9%), Reb C(2.4%), Reb D (0.1%), Reb B (0.1%), Reb E (0.3%), Reb F (0.2%),Steviolbioside (0.1%), and Dulcoside A (0.3%) was 48.0% from initialmaterial, and the content of total steviol glycosides was 97.3% on a drybasis.

The filtrate and washing liquid were combined and spray dried. Thecontent of steviol glycosides was as follows: Stevioside (3.8%), Reb A(56.3%), Reb C (25.9%), Reb D (4.8%), Reb B (0.4%), Reb E (1.6%), Reb F(3.00/%), Steviolbioside (0.1%), and Dulcoside A (4.0%). The yield ofthe product was around 299 g.

The isolation and purification of Reb D from the spray dried remainingsolution was carried out using a method similar to that described inEXAMPLE 5.

The yield of precipitate with 99.3% of Reb D contents was 9.4 g.

Example 11: Purified Steviol Glycoside Mixture from Stevia rebaudianaBertoni Plant Leaves

2.5 kg of Stevia rebaudiana Bertoni dried plant leaves containingStevioside—2.6%, Reb A—6.9%, Reb B—0.01%, Reb C—1.1%, Reb D—0.05%, RebE—0.03%, Reb F—0.3%, Dulcoside A—0.2%, Rubusoside 0.1% andSteviolbioside—0.01% were extracted with 50.0 L of water at a pH of 6.5at 40° C. for 160 min. The biomass was separated by filtration, 500 gcalcium oxide was added to the filtrate to adjust the pH within therange of 8.5-9.0, and the mixture was maintained for 15 min with slowagitation. The pH was then adjusted to around 3.0 by adding 750 g ofFeCl₃, and the mixture was maintained for 15 min with slow agitation. Asmall amount of calcium oxide was further added to adjust the pH to8.5-9.0 and the mixture was maintained for 30 min with slow agitation.The precipitate was removed by filtration and the filtrate was passedthrough 4 columns, each packed with 1.5 liters of specific macroporouspolymeric adsorbent YWD-03 (Cangzhou Yuanwei, China) at BV 1.0 hour⁻¹.Further, the resin sequentially was washed with 1 volume of water, 2volumes of 0.5% NaOH, 1 volume of water, 2 volumes of 0.5% HCl, andfinally with water until the pH was 7.0. Desorption of the adsorbedsteviol glycosides was carried out with 52% ethanol at SV=1.0 hour⁻¹.Desorption of the last column was carried out separately, and thefiltrate was not mixed with the main solution obtained from the othercolumns. First 20 L of eluate from the first three columns was collectedand mixed with 0.3% of activated carbon from the total volume ofsolution. The suspension was maintained at 25° C. for 30 min withcontinuous agitation. Separation of carbon was carried out on apress-filtration system. The obtained filtrate was passed through thecolumns packed with cation-exchange resin Amberlite FCP22 (H⁺) followedwith anion-exchange resin Amberlite FPA53 A30B (OH⁻). The flow rate inboth columns was around SV=0.5 hour-1. The ethanol was distilled using avacuum evaporator. The solids content in the final solution was around15%. The concentrate was passed through the columns packed with 200 mLof LX-18 macroporous adsorbent resin (Xi'an SunResin Technology Ltd.,China). A solution was obtained after the column was passed throughcation-exchange resin Amberlite FCP22 (H⁺) and anion-exchange resinAmberlite FPA53 (OH⁻) with SV=0.5 hour⁻¹. The resulting solution wasconcentrated by a nano-filtration device to around 50% of solidscontent, and spray dried to obtain a highly purified mixture of steviolglycosides. The yield was 150.1 g with 97.5% TSG content, comprisingStevioside—22.4%, Reb A—60.0%, Reb B—0.1%, Reb C—9.1%, Reb D—0.4%, RebE—0.3%, Reb F—2.6%, Dulcoside A—1.5%, Rubusoside 1.0% andSteviolbioside—0.1%.

Example 12: Purified Steviol Glycoside Mixture from Low Purity SteviolGlycoside Mixture

Six kg of steviol glycoside mixture with a TSG content of 85.29% (w/w ona dry basis), comprising 0.16% Rubusoside, 0.68% Dulcoside A, 26.20%Stevioside, 9.78% Rebaudioside C, 1.86% Rebaudioside F, 45.92%Rebaudioside A, 0.51% Rebaudioside D, 0.09% Steviolbioside, and 0.09%Rebaudioside B, was dissolved in 114 L of water.

The solution was passed through 6 consecutively connected columns, eachpacked with 20 L of YWD-03 (Cangzhou Yuanwei, China) macroporous resinat 1.0 BV per hour.

The columns were then washed consecutively with 200 liters of water, 120liters of 0.5% NaOH, 200 liters of water, 120 liters of 0.5% HCl, 360liters of water, and 120 liters of 10% aqueous ethanol.

The columns from the second column through the fifth column weredisconnected and the adsorbed steviol glycosides were eluted with 40liters of 50% aqueous ethanol. The obtained solution was evaporated,concentrated and spray dried to yield 2.5 kg purified steviol glycosidemixture with a TSG content of 98.56% (w/w on a dry basis) comprising0.18% Rubusoside, 0.78% Dulcoside A, 30.31% Stevioside, 11.31%Rebaudioside C, 2.15% Rebaudioside F, 53.04% Rebaudioside A, 0.59%Rebaudioside D, 0.10% Steviolbioside, and 0.10% Rebaudioside B.

Example 13: Low-Calorie Orange Juice Drink with Reb D

60 g of concentrated orange juice were mixed with 1.1 g of citric acid,0.24 g of vitamin C, 1.0 g of orange essence, 0.76 g of Reb D and water,to create a homogeneously dissolved mixture of 1000 mL, in total amount.Then, the mixture was sterilized for a period of 20 seconds at about 95°C. in order to prepare an orange juice similar to one made byconventional methods. The product had an excellent taste profile.

Juices from other fruits, such as apple, lemon, apricot, cherry,pineapple, etc. can be prepared using the same approach.

Example 14: Low-Calorie Carbonated Lemon-Flavored Beverage with Reb D

Sugar (30.0 kg), citric acid (2.5 kg), green tea extract (25.0 kg), salt(0.3 kg), lemon tincture (10.0 L), juniper tincture (8.0 L), sodiumbenzoate (0.17 kg) and Reb D (0.4 kg) were mixed well and dissolved incarbonated water making 1000 L of a mixture.

Example 15: Low-Calorie Carbonated Drink with Reb D

The formula for the beverage is provided in TABLE 67.

TABLE 67 Ingredients Quantity, % Cola flavor 0.340 Phosphoric acid (85%)0.100 Sodium citrate 0.310 Sodium benzoate 0.018 Citric acid 0.018Sweetener 0.030 Carbonated water to 100

Example 16: Chocolate with Reb D

A composition containing 30 kg of cacao liquor, 11.5 kg of cacao butter,14 kg of milk powder, 44 kg of sorbitol, 0.1 kg of salt, and 0.1 kg ofhigh purity Reb D was kneaded sufficiently, and the mixture was thenplaced in a refiner to reduce its particle size for 24 hours.Thereafter, the content was transferred into a conche, 300 grams oflecithin was added, and the composition was kneaded at 50° C. for 48hours. Then, the content was placed in a shaping apparatus, andsolidified.

The product is a low-cariogenic and low-calorie chocolate, withexcellent texture and sensory characteristics.

Example 17: Ice Cream with Reb D

1.50 kg of whole milk was heated to 45° C., and 300 grams of milk cream,100 grams of tagatose, 90 grams of sorbitol, 6 grams of carrageenan as astabilizer, 3 grams of polysorbate-80 as an emulsifier, and 1.0 gram ofReb D as in EXAMPLE 10, were added into the milk and stirred until theingredients completely dissolved. The mixture then was pasteurized at atemperature of 80° C. for 25 seconds. The homogenization of the obtainedmixture was carried out at a pressure of 800 bars and the samples werekept at a temperature of 4° C. for 24 hours to complete the agingprocess. Vanilla flavor (1.0% of the mixture weight) and coloring(0.025% of the mixture weight) were added into the mixture after aging.The mixture was then transferred to an ice cream maker to produce icecream automatically. Samples of produced ice creams were transferred tosealed containers and were kept in the freezer at a temperature of −18°C.

The application of sweeteners does not affect the physicochemicalproperties of ice cream, as well as the overall attributes of color,smoothness, surface texture, air cell, vanilla aroma intensity, vanillataste, chalkiness, iciness and melting rate.

Example 18: Yoghurt with Reb D

In 5 kg of defatted milk, 4.0 grams of high purity Reb D preparedaccording to the invention was dissolved. After pasteurizing at 82° C.for 20 minutes, the milk was cooled to 40° C. A starter in the amount of150 grams was added and the mixture was incubated at 37° C. for 6 hours.Then, the fermented mass was maintained at 10-15° C. for 12 hours.

The product is a low-calorie and low-cariogenic yoghurt, without foreigntaste and odor.

Example 19: Tabletop Tablets with Reb D

A mixture, consisting of 58.5% lactose, 10% calcium silicate, 5%cross-carmellose, 5% L-leucine, 1% aerosil 200, 0.5% magnesium stearate,and 20% of high purity Reb D, obtained according to the invention frominitial Stevia extract or remaining solution after isolation of Reb Aand steviol glycosides, was kneaded sufficiently. Then the mixture wasshaped with the use of a tabletting machine, equipped with punches of6.2 mm diameter, into tablets of 70 mg each, 3.0 mm thick, and 10±1 kghardness.

The tablets can be easily administered due to their favorable sweetnessand fast solubility.

Example 20: Iced Lemon Tea Sweetened with Reb D

The formula for the beverage is provided in TABLE 68.

TABLE 68 Ingredients Quantity, % High purity Reb D 0.08 Sodium benzoate0.02 Citric acid 0.27 Ascorbic acid 0.01 Tea extract 0.03 Lemon flavor0.10 Water to 100

All ingredients were blended and dissolved in the water, andpasteurized. The product possessed excellent taste and flavor.

Example 21: Bread with Reb D

1 kg of flour, 37.38 grams of fructooligosaccharide syrup, 80 grams ofmargarine, 20 grams of salt, 20 grams of yeast, and 0.25 grams of highpurity Reb D obtained according to the invention were placed into theblender and mixed well. 600 mL of water was poured into the mixture andkneaded sufficiently. At the completion of the kneading process, thedough was shaped and raised for 30 to 45 minutes. The raised dough wasplaced in an oven and baked for 45 minutes. Bread samples had a creamywhite color and smooth texture.

Example 22: Diet Cookie with Reb D

Flour, 50.0%; margarine, 30.0%; fructose, 10.0%; maltitol, 8.0%; wholemilk, 1.0%; salt, 0.2%; baking powder, 0.15%; vanillin, 0.1%; and Reb D,0.55%, obtained according to this invention, were kneaded well in adough-mixing machine. After molding of the dough the cookies were bakedat 200° C. for 15 minutes.

The product is a low-calorie diet cookie with excellent properties andappropriate sweetness.

Example 23: Cake with Reb D

123 g of hen eggs, 45 g of sugar, 345 g of sorbitol liquid, 2.0 g ofsucrose fatty acid ester, and 0.35 g of Reb D were mixed with 100 g ofwheat flour and with 200 g of water in order to prepare a cake accordingto a common method. The product had an excellent taste with an optimalsweet flavor.

Example 24: Marshmallow with Reb D

100 g of egg whites were mixed with 280 g of sugar and after foaming wasinduced, the product was mixed with 480 g of gelatin and 144 g of waterto create a homogeneous gelatin solution. Next, 126 g of 70% sorbitolliquid was added, as well as 1.7 g of Reb D and a small essence amountso that 1.13 kg of marshmallow was manufactured according to aconventional method. The product had an excellent taste with a sweetflavor.

Example 25: Soy Sauce with Reb D

0.8 g of Reb D was added to 1000 mL of soy sauce and mixed homogenously.The product had an excellent taste as well as a stable product quality.

Example 26: Tooth Paste with Reb D

A tooth paste was prepared by kneading a composition comprising calciumphosphate, 45.0%; carboxymethylcellulose, 1.5%; carrageenan, 0.5%;Glycerol, 18.0%; polyoxyethylene sorbitan mono-ester, 2.0%;beta-cyclodextrin, 1.5%; sodium laurylsarcosinate, 0.2%; flavoring,1.0%; preservative, 0.1%; Reb D, obtained according to this invention,0.2%; and water to 100%, in a conventional manner. The product possessedexcellent foaming and cleaning abilities with excellent sweetness.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

We claim:
 1. A method for purifying steviol glycosides, comprising thesteps of: a. providing a solution of steviol glycosides; b. passing thesolution of steviol glycosides through a multi-column system including aplurality of columns packed with an adsorbent resin, to obtain at leastone column having adsorbed steviol glycosides; c. removing impuritiesfrom the multi-column system; and d. eluting the adsorbed steviolglycosides from the at least one column, to obtain an eluted solutioncomprising steviol glycosides.
 2. The method of claim 1, whereinimpurities are removed by eluting the multi-column system with a washingsolution.
 3. The method of claim 2, wherein the washing solutioncomprises an aqueous alcohol solution having a water to alcohol ratioranging from about 99.9:0.1 to about 60:40.
 4. The method of claim 1,wherein the adsorbed steviol glycosides are eluted with an aqueousalcohol solution having a water to alcohol ratio ranging from about60:40 to about 0.1:99.9.
 5. The method of claim 1, wherein at least twocolumns of the plurality of columns are connected in series.
 6. Themethod of claim 1, wherein at least two columns of the plurality ofcolumns are connected in parallel.
 7. The method of claim 1, wherein aratio of a volume of a first column of the plurality of columns to avolume of a second column of the plurality of columns is in the range ofabout 1:1 to about 1:10.
 8. The method of claim 1, wherein a ratio of avolume of a last column of the plurality of columns to a volume of apenultimate column of the plurality of columns is in the range of about3:1 to about 1:10.
 9. The method of claim 1, wherein the adsorbent resincomprises a macroporous polymeric resin capable of adsorbing steviolglycosides.
 10. The method of claim 1, wherein the solution of steviolglycosides is obtained by dissolving a steviol glycoside mixture in asolvent, and further comprising the step of drying the eluted solutioncomprising steviol glycosides to obtain a purified steviol glycosidemixture.
 11. The method of claim 10, wherein the purified steviolglycoside mixture has a purity level of at least about 95% (w/w) on adry basis.
 12. A purified steviol glycoside mixture made by the methodof claim
 10. 13. A product comprising the purified steviol glycosidemixture of claim 12, wherein the product is selected from the groupconsisting of a food, a beverage, a pharmaceutical composition, atobacco product, a nutraceutical composition, an oral hygienecomposition, and a cosmetic composition.
 14. The method of claim 1,wherein the solution of steviol glycosides is obtained by dissolving asteviol glycoside mixture in a solvent, and wherein prior to elutingadsorbed steviol glycosides, high Rebaudioside D fractions are elutedwith an aqueous alcohol solution.
 15. The method of claim 14, furthercomprising the steps of: a. mixing the high Rebaudioside D fractionswith a first aqueous alcohol solution to obtain a Rebaudioside Dsolution; b. inducing crystallization to obtain first crystals ofRebaudioside D; c. separating the first crystals of Rebaudioside D fromthe Rebaudioside D solution, said first crystals having a purity levelgreater than about 60% (w/w) on a dry basis; d. suspending the firstcrystals of Rebaudioside D in a second aqueous alcohol solution toobtain second crystals of Rebaudioside D and a third aqueous alcoholsolution; and e. separating the second crystals of Rebaudioside D fromthe third aqueous alcohol solution, said second crystals having a puritylevel greater than about 95% (w/w) on a dry basis.
 16. A purifiedRebaudioside D made by the method of claim
 15. 17. A product comprisingthe purified Rebaudioside D of claim 16, wherein the product is selectedfrom the group consisting of a food, a beverage, a pharmaceuticalcomposition, a tobacco product, a nutraceutical composition, an oralhygiene composition, and a cosmetic composition.
 18. The method of claim1, wherein the solution of steviol glycosides is prepared by a.providing a biomass of a Stevia rebaudiana Bertoni plant; b. producing acrude extract by contacting the biomass with water; c. separatinginsoluble material from the crude extract to obtain a filtratecontaining steviol glycosides; and d. treating the filtrate to removehigh molecular weight compounds and insoluble particles to obtain thesolution of steviol glycosides.
 19. The method of claim 18, furthercomprising the steps of: a. treating the eluted solution to obtain adecolorized solution; b. evaporating the solvent from the decolorizedsolution to obtain a first adsorption solution; c. passing the firstadsorption solution through a column with a macroporous adsorbent toobtain a second adsorption solution; d. deionizing and concentrating thesecond adsorption solution; and e. drying the deionized and concentratedsecond adsorption solution to obtain a purified steviol glycosidemixture with at least about 95% by weight total steviol glycosides. 20.A purified steviol glycoside mixture made by the method of claim
 19. 21.A product comprising the purified steviol glycoside mixture of claim 20,wherein the product is selected from the group consisting of a food, abeverage, a pharmaceutical composition, a tobacco product, anutraceutical composition, an oral hygiene composition, and a cosmeticcomposition.
 22. The method of claim 18, further comprising eluting highRebaudioside D fractions from the multi-column system to obtain aneluted Rebaudioside D solution.
 23. The method of claim 22, furthercomprising the steps of: a. treating the eluted Rebaudioside D solutionto obtain a decolorized solution; b. evaporating solvent from thedecolorized solution to obtain a high Rebaudioside D content mixture; c.combining the high Rebaudioside D content mixture with solvent to form aRebaudioside D solution or suspension; and d. crystallizing RebaudiosideD from the Rebaudioside D solution or suspension to obtain RebaudiosideD crystals.
 24. The method of claim 23, further comprising the steps of:a. separating the Rebaudioside D crystals from the Rebaudioside Dsolution or suspension; b. combining the Rebaudioside D crystals withsolvent to form a purified Rebaudioside D solution or suspension; c.crystallizing purified Rebaudioside D from the purified Rebaudioside Dsolution or suspension to obtain purified Rebaudioside D crystals; d.separating the purified Rebaudioside D crystals from the purifiedRebaudioside D solution or suspension; and e. drying the purifiedRebaudioside D crystals.
 25. The method of claim 24, wherein thepurified Rebaudioside D crystals have a purity level greater than about95% (w/w) on a dry basis.
 26. A purified Rebaudioside D made by themethod of claim
 25. 27. A product comprising the purified Rebaudioside Dof claim 26, wherein the product is selected from the group consistingof a food, a beverage, a pharmaceutical composition, a tobacco product,a nutraceutical composition, an oral hygiene composition, and a cosmeticcomposition.